Computer design, synthesis, and bioactivity analyses of drugs like fingolimod used in the treatment of multiple sclerosis

Article abstract of DOI:10.1016/j.bmc.2016.11.015

Multiple sclerosis (MS) is a very common disease of vital importance. In the MS treatment, some drugs such as fingolimod which help to protect nerves from damage are used. The main goal of the drug therapy in MS is to take control of the inflammation whic

Full text of DOI:10.1016/j.bmc.2016.11.015

Accepted Manuscript
Computer design, synthesis, and bioactivity analyses of drugs like Fingolimod
used in the treatment of Multiple Sclerosis
Gurbet Çelik Turgut, Doğukan Doyduk, Yılmaz Yıldırır, Serkan Yavuz, Atilla
Akdemir, Ali Dişli, Alaaddin Şen
PII:
S0968-0896(16)31168-3
DOI:
http://dx.doi.org/10.1016/j.bmc.2016.11.015
BMC 13382
Reference:
Bioorganic & Medicinal Chemistry
To appear in:
Received Date:
Revised Date:
Accepted Date:
1 August 2016
2 November 2016
9 November 2016
Please cite this article as: Çelik Turgut, G., Doyduk, D., Yıldırır, Y., Yavuz, S., Akdemir, A., Dişli, A., Şen, A.,
Computer design, synthesis, and bioactivity analyses of drugs like Fingolimod used in the treatment of Multiple
Sclerosis, Bioorganic & Medicinal Chemistry (2016), doi: http://dx.doi.org/10.1016/j.bmc.2016.11.015
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Computer design, synthesis, and bioactivity analyses of drugs like Fingolimod used in
the treatment of Multiple Sclerosis
Gurbet Çelik Turgut a, Doğukan Doyduk b, Yılmaz Yıldırır b,*, Serkan Yavuz b, Atilla Akdemir c,
Ali Dişli b, Alaaddin Şena
a
Department of Biology, Faculty of Arts & Sciences, Pamukkale University, 20070 Kınıklı, Denizli,
Turkey
b
Department of Chemistry, Faculty of Science, Gazi University, 06500, Ankara, Turkey
c
Deptment of Pharmacology, Faculty of Pharmacy, Bezmialem Vakıf University, 34093 İstanbul,
Turkey
Abstract
Multiple Sclerosis (MS) is a very common disease of vital importance. In the MS treatment,
some drugs such as fingolimod which help to protect nerves from damage are used. The
main goal of the drug therapy in MS is to take control of the inflammation which leads to the
destruction of myelin and axons in nerve cell and thus prevent and stop the progression of
the disease. Fingolimod (FTY720) is an orally active immunomodulatory drug that has been
used for the treatment of relapsing-remitting multiple sclerosis. It is a sphingosine-1-
phosphate receptor modulator which prevents lymphocytes from contributing to an
autoimmune reaction by inhibiting egress of lymphocytes them from lymph nodes. In this
study, we have computer designed, synthesized and characterized two novel derivatives of
FTY720, F1-12h and F2-9, and have determined their underlying mechanism of their
beneficial effect in SH-SY5Y, SK-N-SH, and U-118MG cell lines. For this purpose, we first
determined the regulation of the cAMP response element (CRE) activity and cAMP
concentration by F1-12h and F2-9 together with FTY720 using pGL4.29 luciferase reporter
assay and cAMP immunoassay, respectively. Then, we have determined their effect on MS-
and GPCR-related gene expression profiles using custom arrays along with FTY720
treatment at non-toxic doses (EC10). It was found that both derivatives significantly activate
CRE and increase cAMP concentration in all three cell lines, indicating that they activate
cAMP pathway through cell surface receptors as FTY720 does. Furthermore, F1-12h and
F2-9 modulate the expression of the pathway related genes that are important in
inflammatory signaling, cAMP signaling pathway, cell migration as well as diverse receptor
and transcription factors. Expression of the genes involved in myelination was also increased
by the treatment with F1-12h and F2-9. In summary, our data demonstrate that the two novel
FTY720 derivatives act as anti-inflammatory ultimately by influencing the gene expression

via the cAMP and downstream transcription factor CRE pathway. In conclusion, F1-12h and
F2-9 might contribute future therapies for autoimmune diseases such as multiple sclerosis.
Key Words: Multiple Sclerosis (MS), Fingolimod, Neuroblastoma, Glioblastoma, S1P1
Agonists, Molecular modeling studies
1. Introduction
Multiple sclerosis (MS) is a chronic autoimmune disease characterized by demyelinated
plaques with gliosis and inflammatory infiltrates that mainly consist of lymphocytes and
mononuclear phagocytes in central nervous system (CNS).1 Although MS was first
recognized in the19th century, the cause of the disease remains a mystery, but it appears to
involve a combination of genetic and non-genetic factors that together result in a self-
sustaining autoimmune disorder that leads to recurrent immune attacks on the CNS. 2,3
MS affects more than 2.3 million people worldwide and mainly young adults with a
predominance of females.4 It is one of the most common neurological disorders but there is
still no complete cure for MS, and it remains entirely non-treatable disease with no effective
treatment. Currently, the goals of MS treatment include decreasing their frequency,
shortening the duration of acute exacerbations and providing symptomatic relief and so Food
and Drug Administration (FDA) approved therapies for MS are not curative1. Fingolimod
(FTY720) was endorsed by the United FDA in September 2010 as the first oral treatment for
MS. FTY720 reduced the number of lesions and clinical disease activity in patients with MS
but has revealed side effects such as associated with an initial reduction in the heart rate and
asymptomatic elevations of alanine aminotransferase.5 Therefore, pharmaceutical
companies and scientists are still searching for new drug candidates that are more effective
than existing drugs and have fewer side effects.
In this context, our aim was the development of structurally novel hS1P1R agonists with a
potential use as therapeutic agents against MS. To this end, a virtual library of synthetically
accessible fingolimod derivatives has been designed. Subsequently, docking studies have
been performed using crystal structures of hS1P1R to identify derivatives that were expected
to show high affinity and these compounds were then synthesized. The chemical syntheses
of F1-12h and F2-9 have been carried to achieve the intended goals. After confirming the
purity and identity of the synthesized product, it was to analyze the impact of FTY720 and its
derivatives on multiple sclerosis and G-Protein-Coupled Receptor (GPCRs) signaling
pathway profiles of SH-SY5Y, SK-N-SH, and U-118 MG cells. Therefore, the data obtained
from these studies have been a help to understand the disease by giving an opportunity for
the identification novel molecular targets.

2. Molecular docking and chemistry
2.1. Docking studies
All ligands were docked into the binding pocket of hS1P1R (3V2W) using the Gold Score
scoring function. The F6 compound series contains a tricyclic moiety that forms steric
clashes with the binding pocket, and the compounds were unable to address the
hydrophobic subpocket.
Compounds of series F1, F3, F4, F5 and F6 all formed similar binding interactions with the
receptor (see figure 2 for representative poses for compounds F1 and F2). Their cationic
amine groups are located near the entrance of the cavity and are able to form hydrogen
bonding interactions with the backbone carbonyl group of Val194. The ligand’s two hydroxyl
substituents can form additional hydrogen bonding interactions with the side chains of Lys34
and Arg120. The distance between the cationic amine of the ligand to the cationic groups of
Lys34 and Arg120 is approximately 5 Å and 3.9 Å, respectively. The hydrophobic tail of the
ligand is able to position itself into the hydrophobic subpocket and form hydrophobic
interactions to Phe125. In general, longer tails showed more hydrophobic interactions with
the subpocket compared to the shorter tails. Halogen or nitro substituents on the para-
position of the F5 series did not form any electrostatic interactions with the residues of the
hydrophobic site. Compounds F1and F2 were selected for bioactivity assays since they
showed the highest docked scores. The binding interactions of the ligand in the two crystal
structures are very similar (Figure 1). The ligand-bound orthosteric binding pockets of the
3V2W and 3V2Y structures of hS1P1R have been superposed (Figure 2).

Lys34
Leu195
Glu121
Val194
Leu276
Phe273
Phe125
Arg120
Glu294
Phe210
Leu272
Tyr98
Leu297
Trp269
Figure 1. The docked pose of compounds F1 (turquiose; GoldScore: 73.1487) and F2 (purple;
GoldScore: 73.5783) in the binding pocket of hS1P1R (3V2W structure). Hydrogen bonds are
indicated with red dashed lines.
Lys34
Val194
Leu276
Lys34
Tyr29
Phe273
Glu121
Phe125
Phe210
Asn101
Leu128
Leu297
Trp269
Figure 2.The ligand-bound orthosteric binding pockets of the 3V2W and 3V2Y structures of hS1P1R
have been superposed. The antagonist ML5 has been depicted in purple (3V2W) and light brown
(3V2Y). For clarity, only the residues of 3V2W are depicted in light grey, except for Lys34 and
Asn101, which are depicted for both crystal structures (3V2Y in black). Hydrogen bonds between the
ligand and residues of the 3V2W structure are indicated in red dashed lines (ligand in purple, residues
in light grey). In addition, two hydrogen bonds between the ligand of 3V2Y (light brown) and Lys34
and Asn101 (black) are also indicated.

2.2. Chemistry
Initially, F1-2 was prepared from cyanoacetamide according to a slightly modified literature
method. The tetrazole ring was constructed by the [3+2] 1,3-dipolar cycloaddition of the azide
anion to cyanoacetamide. The alkylation reaction of F1-2 with octylbromides gave the
corresponding N-1 alkylated tetrazoles in good yields. Ethyl ester F1-2 was reduced to the
corresponding alcohol F1-3 in sodium borohydride-methanol system. After conversion of
alcohol into iodides F1-6, condensation of F1-6 and N-protected diethyl aminomalonate gave
diester, F1-7. The ester groups of F1-7 were reduced to hydroxymethyl groups, which were
subsequently protected to give the target compound.
The
preparation
of
2-amino-2-(2-(1-octyl-1H-tetrazol-5-yl)ethyl)propane-1,3-diol
hydrochloride (F1-12h) is outlined in Scheme 1. The synthetic procedure for the 3-amino-3-
(hydroxymethyl)-1-(4-(1-octyl-1H-tetrazol-5-yl)phenyl)butane-1,4-diol hydrochloride (F2-9h) is
shown in Scheme 2.

Scheme 1. Synthesis of 2-amino-2-(2-(1-octyl-1H-tetrazol-5-yl)ethyl)propane-1,3-diol
hydrochloride (F1-12h).

Scheme
2.
Synthesis
of
3-amino-3-(hydroxymethyl)-1-(4-(1-octyl-1H-tetrazol-5-
yl)phenyl)butane-1,4-diol hydrochloride (F2-9).
3. Result and discussion
3.1. Consideration of protein structures for docking studies
Two crystal structures of hS1P1R, both in complexes with the sphingolipid mimic antagonist
ML5, are available from the PDB server (3V2W and 3V2Y). The Cα-atoms of both proteins
superpose well (RMSD: 0.404 Å over 442 residues), and the overall protein fold is very
similar. In addition, the residues of the binding pocket overlap well and only two residues
have different conformations between the two crystal structures (Lys34 and Asn101).
However, they still interact with the cocrystallized antagonist.
The binding interactions of the ligand in the two crystal structures are very similar (Figure 1).
The phosphate group of the ligand forms hydrogen bonds with the side chains of Tyr29 and
+
Lys34 as well as electrostatic interactions with the cationic Lys34 and Arg120. The –NH3
group of the ligand forms hydrogen bonds with the side chains of Asn101 and Glu121.
Finally, the aromatic moiety and aliphatic tail of the ligands are positioned similarly and
interact with the hydrophobic subpocket formed by Phe125, Leu128, Phe210, Leu272,
Leu276 and Leu297. Lys34 and Asn101 have different conformations between the two
crystal structures, but they are able to make similar interactions to the ligand.

For docking studies, the 3V2W structure was selected because Lys34 is further away from
the binding pocket and is expected to exert less repulsive forces on the ligands positive
amine group.
3.2. Biological activities
Currently, there are FDA-approved several drugs for the treatment of MS, but these drugs
are not a complete cure for the disease. However, substantial improvements have been
made towards the treatment of this disease in recent years. One such significant advance is
the first oral agents used to treat the disease. Fingolimod is a first approved oral therapy for
multiple sclerosis. It has high efficiency for MS treatment but constitutes a very adverse
effect5. Therefore, pharmaceutical companies and scientists are still searching for new drug
candidates that are more effective than existing drugs and have fewer side effects. In this
context, the present study investigates the mechanism of action of two novel FTY720
derivatives, F1-12h, and F2-9, together with FTY720 in SK-N-SH, SH-SY5Y, and U-118 MG
cells.
For this purpose, it was primarily aimed to determine the non-toxic doses. In preclinical
toxicology studies, EC10 (the dose lethal to 10% of cell treated) represents a safe phase I
trial starting dose and EC values for humans are best estimated using human cell cultures.9
Therefore, EC10 dose for FTY720, F1-12h, and F2-9 in human neuroblastoma and
glioblastoma cells were investigated by crystal violet staining. Figure 3 showed that cell
viability with ranging from 0 to 20 µM with FTY720, F1-12h, and F2-9 in cell lines. EC10
values for FTY720, F1-12h and F2-9 were calculated 0.55 µM, 0.37 µM and 0.27 in SK-N-
SH; 0.54 µM, 0.45 µM and 0.30 µM in SH-SY5Y; 0.61 µM, 0.54 µM and 0.36 µM in U-118
MG; respectively, at 24h (Table1). All cell lines appeared to be more resistant to FTY720 and
more susceptible to F1-12h and F2-9.

Figure 3: Effect of FTY720, F1-12h and F2-9 on A) SK-N-SH B) SH-SY5Y and C) U-118 MG
cells viability. Cell cultures were treated with increasing concentrations of FTY720 and its
derivatives for 24 hours and cell viability was determined by the crystal violet staining.
Results are expressed as means of two independent experiments, with each experiment run
in triplicate.
Table 1: EC10 values of FTY720 and its derivatives in SH-SY5Y, SK-N-SH and U-188 mg
Cell lines
EC10 (µM) SH-SY5Y
SK-N-SH
U-118 MG
FTY720 and its
derivatives
FTY720
F1-12h
F2-9
0,54
0,45
0,30
0,55
0,37
0,27
0,61
0,54
0,36
After determining the EC10 in cell lines for the compounds, the CRE-reporter and cAMP
concentration was measured in cultured cells with the FTY720, F1-12h, and F2-9 treatment
to monitor the activity of cAMP/PKA signal transduction pathways. We have determined that
both FTY720 derivatives activate the CRE, which is the main point of convergence for
extracellular signals through GPCR using cAMP, as does FTY72010-12. We have
experimentally confirmed the binding of F1-12h and F2-9 to the GPRC using CRE-
responsive luciferase construct in HEK-293 cell lines (Figure 4). Also, increments displayed
in intracellular cAMP concentrations after F1-12h and F2-9 treatments demonstrated their
FTY720 agonist action through cell surface receptors.

Figure 2: (A) cAMP concentration level after forskolin (control), FTY720, F1-12h and F2-9
treatment in SH-SY5Y, SK-N-SH and U-118 MG cells. (B) HEK-293 cells transfected with
CRE firefly luciferase (Luc) construct were treated with forskolin (control), FTY720, F1-12h
and F2-9 for the time indicated and luciferase activities were measured. CRE-dependent
promoter activities are presented in RLUs (defined in Materials and Methods) relative.

Results of three independent experiments with duplicate measurements normalized to hRluc
activities.
Figure 4. (A) cAMP concentration level after forskolin (control), FTY720, F1-12h and F2-9
treatment in SH-SY5Y, SK-N-SH and U-118 MG cells. (B) HEK-293 cells transfected with
CRE firefly luciferase (Luc) construct were treated with forskolin (control), FTY720, F1-12h

and F2-9 for the time indicated and luciferase activities were measured. CRE-dependent
promoter activities are presented in RLUs (defined in Materials and Methods) relative.
Results of three independent experiments with duplicate measurements normalized to hRluc
activities.
In order to further understand the mechanism of action of F1-12h and F2-9, the changes in
the expression level of genes crucial to the MS and GPCR was examined with FTY720, F1-
12h and F2-9 treatment as compared to control cells. Consequently, qPCR custom array
technology was used for this experiment. MS array contained primers for myelination, T-cell
activation/signaling, adaptive immunity, inflammation regulation and response, neuronal and
general apoptosis, cell adhesion, cellular stress, receptors, and transcription factors. GPCR
array includes bioactive lipid receptors, metabotropic glutamate receptors (mGluRs), GABA-
B-like receptors, rhodopsin-like receptors, and secretin-like receptors. Members of the
intricate network of intracellular signaling pathways involved in GPCR-mediated signal
transduction are also represented on this array including the cAMP/PKA Pathway,
Calcium/PKC Pathway, Calcium/NFAT Pathway, PLC Pathway, Protein Tyrosine Kinase
Pathway, PKC/MEK Pathway, p43/p44MAP Pathway, p38/MAP Pathway, PI-3 Kinase
Pathway, NO-cGMP Pathway, Rho Pathway, NFκB Pathway and Jak/Stat Pathway.
Statistically, significant changes in the expression of 55, 52 and 29 genes were detected in
FTY720, F1-12h, and F2-9 treatment, respectively (Table 2 and Supplementary Figure 1 &
2). The mRNA expression levels of different genes from the cell were calculated after
normalizing cycle thresholds against the ‘housekeeping’ gene beta actin (ACTB) and are
presented as the fold change value (2−ΔΔCt) relative to acontrol cell. The altered expression
was observed in genes belonging to all of thegroups mentioned above and involved in
various cellular processes.

Table 2. Expression level of the selected genes in the FTY720, F3A1-3h and F4-9 treatment groups in SH.
groups in SH-SY5Y, SK-N-SH, U-118 MG cells.
SH-SY5Y
U118MG
F3A1-
SKNSH
F3A1-
Myelination/Demyelination
FTY720 F3A1-3h F4-9
FTY720 3h
F4-9
FTY720 3h
F4-9
MAG
4.3772
2.631
-
MAG
4.0174
3.7859 MAG
-
3.5508
3.9862
-
PMP22
PLP1
-5.1694
-3.5921
-3.0787
3.0092 PMP22
3.7859 PLP1
-
-5.8716 -5.1248 5.4523 PMP22
-3.3914 -4.1464 5.2416
3.4958 PLP1
4.9417
-
3.3929
SOD1
3.4032 SOD1
5.2803 SOD1
-
Inflammation/Chemokine/Cytokine
-
-
-
CCL2
-7.8218
4.1393 CCL2
-
2.4869 CCL3
-5.2826
-3.4017
5.2393 CCL2
-
3.7241 CCL3
11.3481 -8.981
4.9933
CCL3
CSF1
-4.8232
-3.1329
-4.0682
-5.716
CSF1
-4.2226
CSF1
-7.7644 -3.004
-
-
-
CXCL10
IFNG
-3.1821
-5.1964
-3.4279
-2.7247
3.1588 CXCL10
-8.4782
-5.5428 -4.5394
4.8547 CXCL10
IFNG
-
-5.2302 -4.7219 6.1156
-6.428 -3.6601
IFNG
-
-
IL10
IL1B
-4.2501
-4.7158
-7.3448
-6.5966
3.3797 IL10
-2.5151 -2.5669 2.6793 IL10
-4.3432 -3.0275 IL1B
-4.2704 -7.7375 4.6028
-5.3866 -4.7796
IL1B
-
-
-
IL6
-6.498
-4.4551
3.0565 IL6
-4.4847 -4.7651 4.3829 IL6
-4.6974 11.9743 -4.899
-
-
-
TNF
-15.9446 -3.2642
-3.511 TNF
-5.8716 -5.8487 6.5178 TNF
15.2356 -7.8456 4.9674
-
-
-
TGFB1
5.0784 TGFB1
5.1404 TGFB1
5.3267
-
-
TGFB2
CCL5
2.8716 TGFB2
-5.915 TGFB2
4.9847
-
-
CCL5
-
CCL5

3.3855
-
5.3124
-
4.1626
CXCL11
-5.0428
-3.9685
CXCL11
-2.9665 4.2262 CXCL11
-3.5538
-
-
CXCL9
IL18
IL13
3.0249 CXCL9
4.1536 CXCL9
4.8484
IL18
IL13
-3.772
-5.9963
IL18
IL13
-5.1137
-6.6894
-
-
IL2
C1S
LTA
-3.9071
-2.6686
-2.54
4.4248 IL2
-4.2107 4.5247 IL2
-4.9224
-5.1582
-5.1941
C1S
LTA
C1S
LTA
Apoptosis
BCL2
FASLG
MAL
RAF1
VEGFA
Cell Adhesion Molecules
-4.2945
-3.0525
-5.5117
-5.0718
-4.3696
-4.8227
BCL2
FASLG
MAL
RAF1
VEGFA
-4.7035 -4.6127
BCL2
FASLG
MAL
RAF1
VEGFA
-2.7547 -7.8184
-3.5415 -2.9626
-4.2778
-5.2666
-7.7644 -7.9138
-3.8671
-3.8172
-4.4402
-6.1896
-7.3035
-
ICAM1
ITGB2
-6.0839
-3.9655
-3.763 ICAM1
-6.8152 -5.127
-4.0523
5.4736 ICAM1
ITGB2
-
-4.0965 -9.5591
-6.3947
ITGB2
-
VCAM1
-4.9933
-6.6923
-5.2706
-5.7901
4.0191 VCAM1
-
3.9092 MMP9
-4.7445 -3.8654 5.7757 VCAM1
-7.0341 -5.4618
-
13.1489 -5.1049
-
MMP9
-6.3039 -4.4691 4.5002 MMP9
Receptors
CD44
EDNRA
-4.0629
6.8804
CD44
EDNRA
CD44
EDNRA
-4.3753
7.6112
3.6859
-6.2326
-6.2787
3.242
4.4344
7.0402
-
EPHA1
-6.442
EPHA1
-3.7679 -4.2116
EPHA1
-3.6983 -7.9001 3.7516
-
-
-
TNFRSF1A
CD4
-10.091
3.2986 TNFRSF1A
3.0144 TNFRSF1A -3.8155 -3.6537 4.1053
CD4
-3.5323
CD4
-
2.5613 HLA-DRB1 -3.4313
TNFRSF1B
-
HLA-DRB1
TNFRSF1B
HLA-DRB1
TNFRSF1B
4.1339
-
-
-

2.9117
3.1207
5.5404
Transcriptional Factors
APP
-10.004
-5.5022
-15.9446
-8.3109
-3.5677
-4.508
APP
-2.5646
APP
-8.0801
-4.444
-6.6778
-3.1808
-2.7499 -7.2696
-8.3361 -4.315
FOXP3
HDAC1
HIF1A
JUN
FOXP3
HDAC1
HIF1A
JUN
FOXP3
HDAC1
HIF1
JUN
MYC
-3.4447
-3.2716
-3.0183 -5.0916
-4.9203 -4.1285
-4.7214
-5.0669
MYC
MYC
-
-
-3.1465 -2.6004 4.6974 NFKB1
-
NFKB1
STAT3
-3.9931
-5.5022
-3.8433
-2.8938
3.5415 NFKB1
-3.6601 -6.9253 3.6999
STAT3
-2.5624
STAT3
ADCY5
-8.0243 -5.0696
cAMP/PKA Signaling
-
-
ADCY5
-5.5117
-4.4924
-5.9067
-6.5966
4.3188 ADCY5
-5.5057 -5.787
-6.08
-
-5.1851 -4.4983 4.6187
-
-
ADORA2A
ADRB2
4.6048 ADORA2A -4.9966 -5.0641 4.4431 ADORA2A -8.1222 -5.915
4.3394
-11.1967 -4.5291
ADRB2
COL1A1
CYP19A1
-4.9171
-5.339
-4.301
ADRB2
COL1A1
CYP19A1
-6.7945 -4.3526
-3.7305 -3.842
-7.2069 -5.5959
COL1A1
CYP19A1
-5.0806
-5.8869
-4.939
-5.8779
-3.3614
-
-
-
GNAS
NOS2
OPRD1
-3.605
-3.9518
5.0193
-8.1531
-4.7071
2.8007
4.6852 GNAS
-2.939
-5.6458 -4.0673
3.818
-2.9212 3.2119 GNAS
-6.1024 -7.631
-5.9976 -5.1582
4.4537
NOS2
OPRD1
NOS2
OPRD1
4.9782
3.2561
-
-
-
S1PR1
TSHR
-8.6788
-4.0629
-4.2848
4.7801
5.0215 S1PR1
-4.1044 -4.1312 3.8312 S1PR1
-5.8741 -4.3001 4.2281
-4.4134 5.2621
TSHR
-3.664
3.1513
TSHR
-
-
-
S1PR3
CRHR1
EDN1
-2.5755
4.2103
7.826
3.7208 S1PR3
4.6278 S1PR3
-4.0052 4.5159
4.6448
7.7711
CRHR1
EDN1
4.2936
5.5464
CRHR1
EDN1
GCGR
-4.2885
GCGR
GCGR
MAPK Signaling
AGT
3.9041
AGT
5.5368
AGT
5.63
FGF2
MAPK1
-8.2392
-3.8516
-2.8875
FGF2
MAPK1
-4.2052 -4.6964
FGF2
MAPK1
-7.3456 -6.7243
-

10.5515
PI3K Signaling
-
AKT1
-9.4316
-4.1766
AKT1
-2.5763 -2.6892
-3.53 -2.9826
AKT1
12.2684 -5.3124
-
11.0378 -3.3101
-5.7333 -7.7108
CFLAR
PIK3CG
-13.7609 -3.1434
-5.6081 -3.5167
CFLAR
PIK3CG
CFLAR
PIK3CG
-3.7491 -4.5938
Glutamate Receptors
GRM1
GRM5
GRM7
GRM2
-20.6061 -4.806
-3.3753
-5.361
GRM1
GRM5
GRM7
GRM2
-6.1329
GRM1
GRM5
GRM7
GRM2
-4.8631 -5.0346
-5.4523
-6.2306
-4.9647
-3.2823
-5.1941
-
GRM4
GRM4
5.0773 GRM4
Rhodopsin Receptors
AGTR2
3.2887
8.9431
AGTR2
4.8347
4.8726
AGTR2
3.8122
9.0827
-
-
DRD2
PTGDR
-9.8151
-4.9331
-3.3881
3.4837 DRD2
-2.6579 -2.5398
-4.4856
DRD2
PTGDR
-4.5217 -3.8822 4.2208
-4.3981
PTGDR

The expressions of pro- and anti-inflammatory chemokines/cytokines such as CCL2, CCL3,
CCL5, CXCL9, CXC10, and CXCL11 have been shown to be prominently increased in MS.13
Analysis of chemokine transcripts revealed that the transcripts for CCL2, CCL3 and CXCL10
were significantly reduced with FTY720 treatment, CCL2, CXCL10 and CXCL11 mRNA
expression significantly decreased with F1-12h treatment. CCL2, CCL3, CCL5, CXCL9 and
CXCL10 mRNA levels were also significantly reduced with F2-9 treatment. Thus, both
FTY720 derivatives suppress the production of inflammatory cytokines/chemokines in these
cell lines. In addition, expressions of IL1B, IL2, IL6, IL10, IL13, IL18, IFNG, TNF, TGFB1,
TGFB2, LTA, CSF and C1S were also expressed in MS patient, which are known to be
significant in the activation of T cells and generation of important mediators of MS.14-17
FTY720 and F1-12h depicted that IL1B, IL6, IL10, TNF, IFNG and CSF expressions were
significantly lowered as well as F1-12h additionally reduced IL2, IL13, IL18, C1S and LTA
mRNA expression levels. F2-9 inhibited only IL6, IL10, TNF, TGFB1 and TGFB2
expressions. Thus, these data strongly suggest that F1-12h is a more potent inhibitor of the
T cell activation and differentiation than parent FTY720 and the other derivative F2-9.
Determinants involved in the leukocyte migration to the CNS are not exactly known, but there
have been significant developments to elucidate the mechanism of leukocyte migration in MS
patient in recent years.18-21 Adhesion and subsequent migration of leukocytes are mediated
by the interaction of integrins on leukocytes with immunoglobulin-like adhesion molecules
such as ICAM-1 and VCAM-1. This study showed that VCAM-1, ICAM-1, MMP9 and EPHA
expressions reduced significantly in all three cell lines with FTY720 and F1-12h treatments,
but F2-9 was not effective in the SK-N-SH cell line. Also, the level of inhibition was more
potent with F1-12h. Therefore, F1-12h may be of vital importance in suppressing T cell
activation and CNS infiltration. Further animal studies are required to evaluate the whether
or not F1-12h may target T cell infiltration into CNS and repress the migration of immune cell
through cell adhesion.
Previous
studies
have
shown
that
the
expression
genes
involved
in
myelination/remyelination such as MAG and PLP are reduced in MS patient whereas SOD
was found altered in relation to age, sex and disease duration in MS patient 22-23. It was
demonstrated that F2-9 was more efficient in increasing the transcripts levels of myelin-
specific proteins such as MAG, PLP1, and PMP22 than both FTY70 and F1-12h. Therefore,
F2-9 may be more beneficial in remyelination of demyelinated axons in MS. Collectively,
these results indicate that FTY720 derivatives were associated with the suppression of

molecules essential for disease development and induction of molecules important in
healing.
In this study, we also investigated the transcription factor genes since they are main
regulators of the transcriptome, controlling differentiation and survival of immune cells.
FTY720 treatment significantly reduced the APP, FOXP3, HDAC1, HIF1A, JUN, MYC,
NFKB1 and STAT3 transcription levels while F1-12h treatment significantly decreased JUN,
MYC, NFKB1, and STAT3. F2-9 did not affect the expressions of above transcription factor
genes. It is suggested that the regulation of transcriptional factors is more likely to be
peculiar to theinflammatory pathway with F1-12h than parent compound FTY720 but needs
further assessment.
A number of studies indicate that GPCRs are critically involved in various aspects of MS
pathogenesis.24 Targeted activation and inactivation of GPCR mediated signaling may
provide novel effective treatment of MS. FTY720, a non-selective S1P1 receptor modulator,
is one of the best examples of targeting GPCRs in MS treatment. All three compound
significantly downregulated S1PR1 but F1-12h and F2-9 also significantly decreased S1PR3
in neuroblastoma and glioblastoma cell lines. Our results suggest that F1-12h and F2-9 are
the non-selective agonists of S1P receptor as FTY720. Furthermore, their glutamate receptor
selectivity changes with the cell lines suggesting that further detailed studies are required to
evaluate their agonist/antagonist roles through GPRCs.
4. Conclusion
According to the results of the bioactive study done, especially one of the synthesized
compounds,
F1-12h
(2-Amino-2-(2-(1-octyl-1H-tetrazol-5-yl)ethyl)propane-1,3-diol
hydrochloride) may represent more effective and selective drug candidate for the treatment
of different autoimmune and inflammatory disease including MS in contrast to FTY720. Thus,
further animal studies will be required to identify its in vivo efficacy and potency.
5.Experimental
5.1. Materials and methods
5.1.1. Chemistry
All the reagents for the syntheses were commercially available and used without further
purification or purified by standard methods prior to use. Melting points were determined
using an Electrothermal 9100 apparatus (Thermo Fisher Scientific, UK), uncorrected. All

NMR spectra were recorded on a Bruker 300 (1H: 300 MHz, 13C: 75 MHz) NMR spectrometer
(Bruker Corporation, Germany), in CDCl3 or DMSO-d6. Chemical shift values were reported
in ppm relative to that for TMS used as an internal reference standard, J in Hz. MS were
obtained from Waters LCT Premier XE LTOF (TOF MS) instruments (Waters Corporation,
Milferd, MA). FTIR spectra were recorded on a Mattson 1000spectrometer (Mattson
Instruments, Baton Rouge, LA) using KBr pellets. The progress of reactions was monitored
by TLC using SilufolUV-254 plates from Merck (Darmstadt, Germany). The products were
isolated by column chromatography on silica gel.
5.2. Synthesis
5.2.1. Synthetic procedure of 2-amino-2-(2-(1-octyl-1H-tetrazol-5-yl)ethyl)propane-1,3-diol
hydrochloride (F1-12h)
5.2.1.1. Synthesis of ethyl 2-(1H-tetrazol-5-yl)acetate (F1-2)
A solution of ethyl cyanoacetate (1 mL, 9.4 mmol), triethylammonium chloride (1.56 g, 11.27
mmol), and sodium azide (0.73 g, 11.27 mmol) in toluene (50 mL) was heated to 100 °C for
16 h. The obtained orange solution with some off -white precipitate was allowed to cool to
ambient temperature and the solvent was removed under vacuum in a rotary evaporator. The
orange residue was dissolved in water and acidified with 3 M hydrochloric acid solution to pH
2. The precipitated solid was filtered and purified by crystallization from hexane-EtOAc
mixture. Mp: 124-126oC [Lit. 124,4oC; 25].
5.2.1.2.Synthesis of ethyl 2-(1-octyl-1H-tetrazol-5-yl] acetate (F1-3)
A mixture of ethyl 2-(1H-tetrazol-5-yl)acetate (F1-2) (1.00 g, 6.41 mmol), anhydrous K2CO3
(1.06 g, 7.69 mmol) and 1-bromooctane (1.48 g, 7.69 mmol) in 30 mL DMF was refluxed for
10 h and the reaction completion was checked by TLC. After refluxing, the reaction mixture
was cooled to room temperature and evaporated under reduced pressure. The residue
product was extracted with ethyl acetate–water (30:10mL). The organic layer was dried over
Na2SO4 and the solvent evaporated. The product purified by CC (hexane/ EtOAc, 6:1). Yield
60%, yellow liquid.26 1H NMR (300 MHz, CDCl3, δ/ppm): 4.59 (t, J=7.2 Hz, 2H), 4.23 (q, J=7.1
Hz, 2H), 3.96 (s, 2H), 2.00 (m, 2H), 1.35 (m, 13H), 0.90 (t, 3H). 13C NMR (75 MHz, CDCl3,
δ/ppm): 168.2 (CO), 159.9 (C), 61.4 (CH2), 53.1 (CH2), 31.8 (CH2), 31.6 (CH2), 29.1 (CH2),
28.9 (CH2), 28.7 (CH2), 26.2 (CH2), 22.5 (CH2), 14.0 (CH3), 13.9 (CH3). IR (ATR, ʋmax/cm-1):
2924, 1738. HRMS(ES): For C13H24N4O2 calcd. 269.1978, found 269.1968.

5.2.1.3. Synthesis of 2-(1-octyl-1H-tetrazol-5-yl)ethane-1-ol (F1-4)
Powdered sodium borohydride (0.85 g, 22.39 mmol) was suspended in THF (30 mL) in the
presence of the ethyl 2-(1-octyl-1H-tetrazol-5-yl] acetate (1.00 g; 3.73 mmol) during a period
of 15 minutes under reflux (70ºC) and stirring. Then methanol (8mL) was added dropwise
during a period of 15 minutes with effervescence being observed. Stirring and reflux were
maintained during a period of 15 to 60 minutes. The reaction completion was checked by
TLC. After the end of the reaction, the alcohol was cooled to room temperature and
quenched with a saturated solution of NH4Cl (10mL) for a further period of 1.5 hours. For
purification of alcohols, the organic layer was separated and the aqueous phase extracted
with ethyl acetate (3x25mL). The organic extracts were combined and dried over Na2SO4 and
concentrated under low pressure to give the alcohol. The product purified by CC (hexane/
EtOAc, 6:1). yield 90%, colorless liquid.27 1H NMR (300 MHz, CDCl3, δ/ppm): 4.57 (t, J=7.2
Hz, 2H), 4.04 (t, 2H), 3.15 (t, J=5.9 Hz, 2H), 2.82 (brs, 1H), 2.00 (m, 2H), 1.25 (m, 10H), 0.90
(t, 3H). 13C NMR (75 MHz, CDCl3, δ/ppm): 163.9 (C), 59.7 (CH2), 52.9 (CH2), 31.4 (CH2),
29.0 (CH2), 28.8 (CH2), 28.6 (CH2), 26.1 (CH2), 22.3 (CH2), 13.7 (CH3). IR (ATR, ʋmax/cm-1):
3368, 2956. HRMS(ES): For C11H22N4O calcd. 227.1872, found 227.1846.
5.2.1.4. 5-(2-Iodoetyl)-1-octyl-1H-tetrazol (F1-6)
Methane sulfonyl chloride (0.76 g, 6.64 mmol) was added to a solution of 2-(1-octyl-1H-
tetrazol-5-yl)etan-1-ol (1.00 g, 4.43 mmol) and triethylamine (0.67 g, 6.64 mmol) in CH2Cl2
(25mL) at 0oC.The suspension was stirred at room temperature for 2h. The reaction mixture
was washed with brine, dried over Na2SO4, and concentrated in vacuum. Sodium iodide
(0,48 g, 3,19 mmol) was added to a solution of the 2-(1-octyl-1H-tetrazol-5-yl)ethyl
methanesulphonate (0,97 g, 3.19 mmol) in 2-butanone (40 mL), and the suspension was
heated under reflux for 5 h. The reaction mixture was diluted with EtOAc and washed with
water, 10% aqueous sodium thiosulfate, and brine. The organic layer was dried over Na2SO4
and concentrated in vacuum. Purification of the residue by silica gel column chromatography
(hexane/EtOAc) gave the title compound. Yield 85%, yellow liquid.28,29 1H NMR (300 MHz,
CDCl3, δ/ppm): 4.60 (t, 2H), 3.51 (m, 4H), 2.00 (m,2H), 1.30 (m, 10H), 0.87 (t, 3H). 13C NMR
(75 MHz, CDCl3, δ/ppm): 164.8 (C), 53.1 (CH2), 31.6 (CH2), 30.9 (CH2), 30.0 (CH2), 29.2
(CH2), 29.1 (CH2), 26.3 (CH2), 22.5 (CH2), 14.0 (CH3). IR (ATR, ʋmax/cm-1): 2954. HRMS(ES):
For C11H21N4I calcd. 337.0889, found 337.0927.

5.2.1.6. 2-Acetamido-2-(2-(1-octyl-1H-tetrazol-5-yl)ethyl)malonate (F1-7)
Powdered sodium hydride (0,89 g, 37,20 mmol) was suspended in THF (30 mL) in presence
of the 5-(2-iodoetyl)-1-octyl-1H-tetrazol (7,50 g, 22,32 mmol) and DEAM (4,85 g, 22,32
mmol) during a period of 15 minutes under reflux (0ºC) and stirring. After refluxing, the
reaction mixture was cooled to room temperature and evaporated under reduced pressure.
The residue product was extracted with ethyl acetate–water (3 x 25mL). The organic layer
was dried over Na2SO4 and concentrated in vacuum. Purification of the residue by silica gel
column chromatography (hexane/EtOAc) gave the acetamido compound. 30 1H NMR (300
MHz, CDCl3, δ/ppm): 6.82 (s, 1H), 4.52 (t, J=7.2 Hz, 2H), 4.24 (m, 4H), 2.82 (m,4H), 2.05 (s,
3H), 1.95 (m, 2H), 1.30 (m, 16H), 0.88 (t, J= 6.97 Hz, 3H). 13C NMR (75 MHz, CDCl3, δ/ppm):
169.2 (CO),167.8 (CO), 165.2 (C), 65.9 (C), 62.7 (CH2), 52.9 (CH2), 31.6 (CH2), 30.6 (CH2),
29.3 (CH2), 29.0 (CH2), 28.8 (CH2), 26.3 (CH2), 22.9 (CH3), 22.6 (CH2), 20.3 (CH2), 14.0
(CH3), 13.9 (CH3). IR (ATR, ʋmax/cm-1): 3302, 2927, 1738, 1680. HRMS(ES): For C20H35N5O5
calcd. 426.2672, found 426.2722.
5.2.1.7.
(F1-9)
N-(1-Hydroxy-2-(hydroxymethyl)-4-(1-octyl-1H-tetrazol-5-yl)butan-2-yl)acetamide
NaBH4 (0.14 g, 3.76 mmol) was added portion wise to a solution of diethyl 2-acetamido-2-(2-
(1-octyl-1H-tetrazol-5-yl)ethyl)malonate (0.40 g, 0.94 mmol) and CaCl2 (0.21 g, 1.88 mmol) in
water (1 mL) and EtOH (5 mL), and stirred at room temperature 16 h. Then 1 M aqueous HCl
was added to the reaction mixture and the mixture was extracted with EtOAc. The organic
layer was washed with brine, dried over Na2SO4, and concentrated in vacuum, yellow
liquid.28
5.2.1.8. 2-Acetamide-2-(2-(1-octyl-1H-tetrazol-5-yl)ethyl)propane-1,3-diyl diacetate (F1-10)
N-(1-Hydroxy-2-(hydroxymethyl)-4-(1-octyl-1H-tetrazol-5-yl)butan-2-yl)acetamide (0.40 g,
1.17 mmol) was dissolved in pyridine (5 mL). Acetic anhydride (0.72 g, 7.04 mmol) was
added to the solution at 0oC. After stirring at room temperature 16 hours, ice and water were
added to the reaction mixture. The mixture was extracted with EtOAc, and the organic layer
was washed successively with water, 0.5 M aqueous HCl, saturated NaHCO3, and brine. The
organic layer was dried over Na2SO4 and concentrated in vacuum, yellow liquid.28.

5.2.1.9. 2-Amino-2-(2-(1-octyl-1H-tetrazol-5-yl)ethyl)propane-1,3-diol (F1-11)
A solution of 2-acetamido-2-(2-(1-octyl-1H-tetrazol-5-yl)ethyl)propane-1,3-diyl diacetate (0,21
g ,0.5 mmol) in MeOH (5.0 mL) and THF (5 mL) was added to a solution of LiOH.H2O (0.11g,
2.5 mmol) in water (5.0 mL), and the mixture was refluxed for 5 hours. After cooling to room
temperature, the reaction mixture was poured into water and extracted with EtOAc. The
organic layer was dried over Na2SO4 and concentrated in vacuum, yellow liquid.28
5.2.1.10. 2-Amino-2-(2-(1-octyl-1H-tetrazol-5-yl)ethyl)propane-1,3-diol hydrochloride(F1-12h)
2-Amino-2-(2-(1-octyl-1H-tetrazol-5-yl)ethyl)propane-1,3-diol (0.15 g, 0.5 mmol) was
dissolved in 4M HCl (5 mL) solution in EtOAc (5 mL) and MeOH (5 mL), and the solvent was
distilled off in vacuum. The yielded white crystalline solid was collected and washed with
EtOAc to give the title compound. Yield 30%, m.p. 83-85oC.28 1H NMR (300 MHz, DMSO-d6,
δ/ppm): 7.96 (s, 1H), 5.42 (t, J=5.0 Hz, 1H), 4.61 (t, J=6.9 Hz, 2H), 3.53 (d, J=5.0 Hz, 2H),
2.93 (t, 2H), 2.01 (t, 2H), 1.86 (m, 2H), 1.25 (m, 10H), 0.85 (t, J= 6.80 Hz, 3H). 13C NMR (75
MHz, DMSO-d6, δ/ppm): 165.9 (C), 61.3 (CH2), 60.4 (C), 52.8 (CH2), 31.6 (CH2), 29.6 (CH2),
29.1 (CH2), 28.9 (CH2), 28.6 (CH2), 26.2 (CH3), 22.5 (CH2), 19.3 (CH2), 14.4 (CH3). IR (ATR,
ʋmax/cm-1): 3344, 3231, 2918. HRMS(ES): Calcd. For C14H29N5O2: 300.2400, found 300.2396.
5.2.2. Synthetic procedure of 3-amino-3-(hydroxymethyl)-1-(4-(1-octyl-1H-tetrazol-5-
yl)phenyl)butan-1,4-diol hydrochloride(F2-9)
5.2.2.1. 1-(4-(1H-Tetrazol-5-yl)phenyl)ethan-1-one (F2-2)
A solution of ethyl 4-acetylbenzonitrile (1 g, 6.84 mmol), triethylammonium chloride (1,13 g,
8,20 mmol), and sodium azide (0.53 g, 8.20 mmol) in toluene (50 mL) was heated to 100 °C
for 16 h. The obtained orange solution with some of-white precipitate was allowed to cool to
ambient temperature and the solvent was removed under vacuum in a rotary evaporator. The
orange residue was dissolved in water and acidified with 3 M hydrochloric acid solution to pH
2. The precipitated solid was filtered and purified by crystallization from hexane-EtOAc
mixture. Yield 81%, mp:190-1920C.31
5.2.2.2.1-(4-(1-Octyl-1H-tetrazol-5-yl)phenyl)ethan-1-one (F2-3)

A mixture of 1-(4-(1H-tetrazol-5-yl)phenyl)etan-1-on (1.00 g, 5.32 mmol), anhydrous K2CO3
(0.88 g, 6.38 mmol) and 1-bromooctane (1.22 g, 6,38 mmol) and in 30 mL DMF was refluxed
for 10 h and the reaction completion was checked by TLC. After refluxing, the reaction
mixture was cooled to room temperature and evaporated under reduced pressure. The
residue product was extracted with ethyl acetate-water (30:10 mL). The organic layer was
dried over Na2SO4 and the solvent evaporated. The product purified by CC (hexane/EtOAc,
6:1), yield 81%, mp: 58-600C.26 1H NMR (300 MHz, CDCl3, δ/ppm): 8.28 (dd, J=6.70 Hz, 2H),
8.10 (dd, J=6.80 Hz, 2H), 4.69 (t, J=7.2 Hz, 2H), 2.63 (s, 3H), 2.11 (m, 2H), 1.35 (m, 10H),
0.89 (t, J= 6,90 Hz , 3H). 13C NMR (75 MHz, CDCl3, δ/ppm): 197.5 (CO), 164.1 (C), 138.2
(C), 131.7 (C), 128.9 (CH), 126.9 (CH), 53.4 (CH2), 31.7 (CH2), 29.4 (CH2), 28.9 (CH2), 28.8
(CH2), 26.7 (CH3), 26.3 (CH2), 22.6 (CH2), 14.0 (CH3). IR (ATR, ʋmax/cm-1): 2987, 2955, 1655.
HRMS(ES): Calcd. For C17H24N4O: 301.202, found 301.2031.
5.2.2.3. 2-Bromo-1-(4-(1-octyl-1H-tetrazol-5-yl)phenyl)ethan-1-one (F2-4)
Solution of Br2 (0.42 g, 2.65 mmol) in CHCl3 (10 mL) was added dropwise to a solution of 1-
o
(4-(1-octyl-1H-tetrazol-5-yl)phenyl)etan-1-on (0.80 g, 2.65 mmol) in CHCl3 (10 mL) at 0 C,
and stirred at room temperature 16 h. Then the mixture was extracted with %10 Na2S2O3 .
The organic layer was washed with brine, dried over Na2SO4, and concentrated in vacuum,
yellow liquid.32 1H NMR (300 MHz, CDCl3, δ/ppm): 8.31 (dd, J=6.80 Hz, 2H), 8.12 (dd, J=6.90
Hz, 2H), 4.69 (t, J=7.20 Hz, 2H), 4.52 (s, 2H), 2.09 (m, 2H), 1.35 (m, 10H), 0.89 (t, J= 6,90
Hz , 3H). 13C NMR (75 MHz, CDCl3, δ/ppm): 190.7 (CO), 163.6 (C), 135.2 (C), 132.5 (C),
129.5 (CH), 127.1 (CH), 53.4 (CH2), 31.7 (CH2), 30.7 (CH2), 29.7 (CH2), 29.4 (CH2), 28.8
(CH2), 26.3 (CH2), 22.6 (CH2), 14.0 (CH3). IR (ATR, ʋmax/cm-1): 2998, 2949, 1701.
HRMS(ES): Calcd. For C17H23BrN4O: 379.1133, found 379.1120.
5.2.2.4. Diethyl 2-acetamido-2-(2-(4-(1-octyl-1H-tetrazol-5-yl)phenyl)-2-oxoethyl)malonate
(F2-5)
Powdered sodium hydride (0,05 g, 2,20 mmol) was suspended in THF (30 mL) in presence
of the 2-bromo-1-(4-(1-octyl-1H-tetrazol-5-yl)phenyl)etan-1-one (0,50 g, 1,32 mmol) and
DEAM (0,48 g, 2,20 mmol) during a period of 15 minutes under reflux (0ºC) and stirring. After
refluxing, the reaction mixture was cooled to room temperature and evaporated under
reduced pressure. The residue product was extracted with EtOAc-water (3 x 25mL). The
organic layer was dried over Na2SO4 and concentrated in vacuum. Purification of the residue
by silica gel column chromatography (hexane/EtOAc) gave the acetamido compound. Yield

60%, yellow liquid.30 1H NMR (300 MHz, CDCl3, δ/ppm): 8.28 (dd, J=8.20 Hz, 2H), 8.10 (dd,
J=8.40 Hz, 2H), 7.14 (s, 1H), 4.69 (t, J=7.20 Hz, 2H), 4.33 (m, 6H), 2.06 (m, 5H), 1.32 (m,
14H), 0.89 (t, J= 7.1 Hz, 3H). 13C NMR (75 MHz, CDCl3, δ/ppm): 196.4 (CO), 169.5 (C),
167.2 (CO), 163.8 (CO), 137.1 (C), 132.3 (C), 128.8 (CH), 127.0 (CH), 64.0 (CH2), 63.0 (C),
53.4 (CH2), 42.4 (CH2),31.7 (CH2), 29.4 (CH2), 29.0 (CH2), 28.8 (CH2), 26.4 (CH2), 23.0
(CH3), 22.6 (CH2), 14.0 (CH3), 13.9 (CH3). IR (ATR, ʋmax/cm-1): 3239, 3048, 2933, 1752,
1737, 1637, 1538. HRMS(ES): Calcd. For C26H37N5O6: 516.2819, found 516.2819.
5.2.2.5.
N-(1,4-dihydroxy-2-(hydroxymethyl)-4-(4-(1-octyl-1H-tetrazol-5-yl)phenyl)butan-2-
yl)acetamide (F2-6):
NaBH4 (0.13 g, 3.48 mmol) was added portionwise to a solution of 2-acetamido-2-(2-(4-(1-
octyl-1H-tetrazol-5-yl)phenyl)-2-oxoethyl)malonate (0.30 g, 0.58 mmol), and CaCl2 (0.19 g,
1,74 mmol) in water (1 mL) and EtOH (5 mL). After stirring at room temperature 16 hours.
Then 1 M aqueous HCl was added to the reaction mixture and the mixture was extracted
with EtOAc. The organic layer was washed with brine, dried over Na2SO4, and concentrated
in vacuum, yellow liquid.28
5.2.2.6.
2-Acetamido-2-(2-acetoxy-2-(4-(1-octyl-1H-tetrazol-5-yl)phenyl)ethyl)propane-1,3-
diyl diacetate (F2-7)
N-(1-hydroxy-2-(hydroxymethyl)-4-(1-octyl-1H-tetrazol-5-yl)butan-2-yl)acetamide(0.25 g, 0.58
mmol) was dissolved in pyridine (5 mL). Acetic anhydride (0.53 g, 5.22 mmol) was added to
the solution at 0 oC. After stirring at room temperature 16 h, ice and water were added to the
reaction mixture. The mixture was extracted with EtOAc, and the organic layer was washed
successively with water, 0.5 M aqueous HCl, saturated NaHCO3, and brine. The organic
layer was dried over Na2SO4 and concentrated in vacuum, yellow liquid.28
5.2.2.7. 3-Amino-3-(hydroxymethyl)-1-(4-(1-octyl-1H-tetrazol-5-yl)phenyl)butan-1,4-diol (F2-
8)
A solution of 2-acetamido-2-(2-acetoxy-2-(4-(1-octyl-1H-tetrazol-5-yl)phenyl)ethyl)propane-
1,3-diyl diacetate (0.32 g, 0.58 mmol) in MeOH (5.0 mL) and THF (5 mL) was added to a
solution of LiOH.H2O (0,16 g, 3,75 mmol) in water (5.0 mL), and the mixture was refluxed for
5 hours. After cooling to room temperature, the reaction mixture was poured into water and
extracted with EtOAc. The organic layer was dried over Na2SO4 and concentrated in vacuum.
Yellow liquid.28

5.2.2.8.
3-Amino-3-(hydroxymethyl)-1-(4-(1-octyl-1H-tetrazol-5-yl)phenyl)butan-1,4-diol
hydrocloride (F2-9)
3-Amino-3-(hydroxymethyl)-1-(4-(1-octyl-1H-tetrazol-5-yl)phenyl)butan-1,4-diol (0.23 g, 0.58
mmol), was dissolved in 4M HCl (5 mL) solution in EtOAc (5 mL) and MeOH (5 mL), and the
solvent was distilled off in vacuum. The yielded white crystalline solid was collected and
washed with EtOAc to give the title compound.28 Yield %17, mp: 2310C. 1H NMR (300 MHz,
DMSO-d6, δ/ppm): 8.04 (dd, J=8.30 Hz, 2H), 7.72 (brs, 1H),7.53 (dd, J=8.30 Hz, 2H), 5.35
(m,1H), 4.97 (m, 1H), 4.72 (t, J=6.90 Hz, 2H), 3.56 (m, 5H), 1.88 (m, 4H), 1.21 (m, 10H),
13
0.83 (t, J= 6,40 Hz , 3H). C NMR (75 MHz, DMSO-d6, δ/ppm): 164.4 (C), 149.1 (C), 126,8
(CH), 126.7 (CH), 126.1 (CH), 68.5 (CH), 62.1 (CH2), 61.6 (C), 60.8 (CH2), 53.2 (CH2), 31.6
(CH2), 29.1 (CH2), 28.9 (CH2), 28.6 (CH2), 26.2 (CH2), 22.5 (CH2), 14.4 (CH3). IR (ATR,
ʋmax/cm-1): 3500-3100, 3052, 2921. HRMS(ES): Calcd. For C20H33N5NaO3: 414.2476, found
414.2471.
5.3. Preparation of ligand molecules
Three-dimensional structures of the synthetically accessible (designed) ligands were
constructed using the MOE program (v2015.10, chemical computing group inc., Montreal,
Canada). All strong acids were deprotonated and all strong bases were protonated.
Afterwards, the ligand structures were energy minimized (steepest-descent, MMFF94x force
field) and saved as a multi-mol2 file.
5.4. Preparation of protein structures
Two crystal structures of the human sphingosine-1-phosphate receptor (hS1P1R) were
obtained from the Brookhaven protein databank, both with the antagonist ML5 complexed to
the orthosteric binding pocket (PDB: 3V2W, 3.35 Å; PDB: 3V2Y, 2.8 Å). Hydrogen atoms
were added to the structures using the “protonate 3D” tool of the MOE software package
(v2015.10, Chemical Computing Group, Inc. Montreal). Afterwards, all buffer and water
molecules were deleted and a steepest-descent energy minimization was performed using
the Amber10:EHT force field. The protein structures were saved as mol2-files.
5.5. Docking studies
All ligand were docked into the orthosteric binding pocket of the 3V2W crystal structure with
the GOLD Suite software package (v5.2, CCDC, Cambridge, UK) and the GoldScore scoring
function. The binding pocket was defined as all residues within 12 Å of the position that

corresponds to the coordinates of the N1 atom of ML5 (x: 3.996; y: 17.198; z: -8.589).
Default settings were used and 50 genetic algorithm runs were performed per ligand.
5.6. Biological studies
5.6.1. Cell Culture
The human neuroblastoma cell lineSH-SY5Y (ATCC CRL-2266), SK-N-SH (ATCC HTB-11),
U-118 MG (ATCC HTB-15) and HEK-293 (ATCC CRL-1573) were purchased from American
Type Culture Collection(USA). SH-SY5Y, SK-N-SH, U-118 MG and HEK-293 was cultured in
DMEM-F12, EMEM, DMEM and EMEM medium, respectively; with 1% antibiotic and
supplemented with 10% heat-inactivated fetal bovine serum in incubated in a humidified
atmosphere with 5% CO2 at 37 °C and was sub-cultured twice a week.
5.6.2. Cell Viability Assay
SH-SY5Y, SK-N-SH, and U-118 MG cells were seeded in 96-well plates at a density of 1x103
cells/ml culture medium. After 24 h incubation, the cells were treated with varying
concentrations (ranging from 0.25 μM to 20 μM) of FTY720, F1-12h, and F2-9. Equal
amounts of medium without FTY720, F1-12h and F2-9 were added to untreated cells
(control). FTY720, F1-12h and F2-9-treated and control cells were incubated for 24 h at 37
°C in humidified 5% CO2 atmosphere. Following incubation, the medium was replaced by
0.5% crystal-violet (w/v; in 50% methanol) solution. Plates were incubated for 10 min at room
temperature, washed with water and adsorbed dye was eluted out with Na-citrate (0.1 M Na-
citrate in 50% ethanol, pH 4.2). Absorbance, which was proportional to cell viability, was
measured at a wavelength of 600 nm. Cell viability was monitored as the percentage of
viable cells compared to control, untreated cells.6
5.6.3. RNA Isolation and cDNA Synthesis
Total RNA from SH-SY5Y, SK-N-SH and U-118 MG was isolated using RNeasy Plus
Universal Mini Kit (Qiagen) according to the manufacturer instruction with slight
modifications. Elution was performed with 50 μL of RNase-free water. Immediately after
elution, RNA concentration was determined using Nanodrop (MaestroNano Micro-volume
Spectrophotometer, USA) and reversely transcribed using Easy Script cDNA Synthesis Kit
(ABM). The reaction mixture was incubated for 50 min at 50 °C followed by termination by

heating at 5 min at 85 °C. cDNA concentration was measured with Nanodrop and distributed
in aliquots before storing at −80°C.
5.6.4. qPCR Quantification Assays
Custom designed human MS and GPRC PCR arrays, containing 160 tested and validated
primers along with three housekeeping genes, and positive and negative controls for RT and
PCR reactions had synthesized from Bioneer and used for quantitative determination of
differential gene expression profiles between different treatment groups. After assuring the
integrity, purity and the quality of cDNA, the whole volume of synthesized cDNA was used for
qPCR protocol. Enough master mix using KiloGreen 2X qPCR Master Mix (ABM, Canada)
were prepared to fill a 96-well plate and recommended thermal cycling parameters on
Exicycler 96 Thermal Block from Bioneer (Korea). PCR Array Data Analysis Web Portal
(Qiagen version 3.5) were applied. Values of cycle threshold (Ct) obtained in quantification
were used for calculations of folds changes in mRNA abundance accordingly to 2−ΔΔCt
method.7 Beta-actin was chosen from the group of three housekeeping genes as the best
and least varying reference gene.
Following determination of the significantly altered genes in FTY720, F1-12h and F2-9-
treated cells as compared to control, further determinations and validations of the identified
genes were performed with the qPCR using custom designed primers listed in
Supplementary Table 1 and 2.
5.6.5. Measurement of cAMP
The cellular cAMP concentration was measured using the cAMP Direct Immunoassay Kit
(BioVision K371; USA). Briefly, the cells were treated with FTY720, F1-12h and F2-9 and the
cells were lysed and processed according to the manufacturer’s protocol using 0.1N HCl.
Afterward, neutralizing buffer and acetylation reagent mixture were added to the cell lysates.
Each acetylated sample and standards were transferred to a protein G-coated 96-well plate.
Rabbit anti-cAMP polyclonal antibody was added to standard and samples, and the reaction
mixtures were incubated for oneꢀhour at room temperature, reincubated with cAMP–HRP for
one hour. The reaction was stopped by adding 1ꢀM HCl and cAMP levels were determined
spectrophotometrically.
5.6.6. CRE-Luciferase assay

To evaluate the transcriptional activity of cAMP, HEK-293 cells were transfected with Fugene
HD reagent (Promega) using pGL4.29 [luc2P/CRE/Hygro] plasmid containing the Renilla
gene used for luciferase normalization. After 24 hour transfection, cells were stimulated with
compounds FTY720, F1-12h, F2-9 and forskolin (positive control) were used. At 18-hour
post stimulations, the cellular lysate was assayed for luciferase and Renilla activities using
the Dual-Glo Luciferase assay system (E2920, Promega). Luminescence was measured
using Synergy HTX luminometer (BioTek). Luciferase activities were normalized with Renilla
activities.8
5.6.7. Statistical analysis
Statistical analyses were performed using the Minitab 13 statistical software package.
(Minitab, Inc., State College, PA, USA). All results were expressed as means including their
Standard Error of Means (SEM). Comparison between groups was performed using
Student’s t-test, and p<0.05 was selected as the level required for statistical significance.
Statistical comparisons between three groups were assessed by one-way analysis of
variance(ANOVA).
Acknowledgements
This work was supported by the Scientific and Technological Research Council of Turkey
[TUBITAK-113Z141] and Pamukkale University PAUBAP-2014FBE028.
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