Geldanamycin-inspired compounds induce direct trans-differentiation of human mesenchymal stem cells to neurons

Article abstract of DOI:10.1016/j.ejmech.2017.03.082

Inspired from geldanamycin, the synthesis of a new series of 20-membered macrocyclic compounds is developed. The key features in our design are (i) retention of the fragment having the precise chiral functional groups of geldanamycin at C10, C11, C12 and C14, and (ii) replacement of an olefin moiety with the ester group, and the quinoid sub-structure with the triazole ring. The southern fragment needed for the macrocyclic ring formation was obtained from Evans' syn aldol as the key reaction and with the use of D-mannitol as the cheap source of a chiral starting material. For the synthesis of the northern fragment, we utilized L-ascorbic acid, which provided the desired chiral functional groups at C6 and C7. Further, the chain extension completed the synthesis of the northern fragment. In our approach, the crucial 20 membered macrocyclic ring was formed employing the click chemistry. When tested for their ability to directly trans-differentiate human mesenchymal stem cells to neurons, two novel compounds (20a and 7) from this series were identified and this was further validated by the presence of specific neuronal biomarkers (i.e. nestin, agrin and RTN4).

Full text of DOI:10.1016/j.ejmech.2017.03.082

Accepted Manuscript
Geldanamycin-inspired compounds induce direct trans-differentiation of human
mesenchymal stem cells to neurons
Srinivas Jogula, Anand Ram Soorneedi, Jagan Gaddam, Srinivas Chamakuri, Girdhar
Singh Deora, Ranjith Kumar Indarapu, Murali Krishna Ramgopal, Subhadra Dravida,
Prabhat Arya
PII:
S0223-5234(17)30248-9
10.1016/j.ejmech.2017.03.082
EJMECH 9342
DOI:
Reference:
To appear in: European Journal of Medicinal Chemistry
Received Date: 6 January 2017
Revised Date: 16 March 2017
Accepted Date: 31 March 2017
Please cite this article as: S. Jogula, A.R. Soorneedi, J. Gaddam, S. Chamakuri, G.S. Deora, R.K.
Indarapu, M.K. Ramgopal, S. Dravida, P. Arya, Geldanamycin-inspired compounds induce direct trans-
differentiation of human mesenchymal stem cells to neurons, European Journal of Medicinal Chemistry
(2017), doi: 10.1016/j.ejmech.2017.03.082.
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Trans-differentiation of human mesenchymal stem cells to neurons by small molecules April 18, 2017
ACCEPTED MANUSCRIPT
Geldanamycin-Inspired Compounds Induce Direct Trans-differentiation of
Human Mesenchymal Stem Cells to Neurons
Srinivas Jogula,† Anand Ram Soorneedi,‡ Jagan Gaddam,† Srinivas Chamakuri, †,$ Girdhar
Singh Deora,# Ranjith Kumar Indarapu,‡ Murali Krishna Ramgopal,‡ Subhadra Dravida,‡,*
and Prabhat Arya†,*
† Dr. Reddy's Institute of Life Sciences (DRILS), University of Hyderabad Campus,
Hyderabad 500046, India
$ Current Address: Baylor College of Medicine, One Baylor Plaza, Houston, TX 77030, USA
‡ Transcell Biologics Pvt. Ltd., ALEAP, Pragathinagar, Hyderabad, Telangana, 500073, India
# School of Pharmacy, The University of Queensland, Brisbane, QLD 4072, Australia
E mail:
suba.dravida@tran-scell.com (SD)
prabhata@drils.org (PA)
Keywords
Human mesenchymal stem cells
Trans-differentiation
Chemical approaches to trans-differentiation
Stem cells to neurons
Natural product-inspired compounds
Geldanamycin
Geldanamycin-inspired compounds
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Trans-differentiation of human mesenchymal stem cells to neurons by small molecules April 18, 2017
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Abstract
Inspired from geldanamycin, the synthesis of a new series of 20-membered macrocyclic
compounds is developed. The key features in our design are (i) retention of the fragment
having the precise chiral functional groups of geldanamycin at C10, C11, C12 and C14, and
(ii) replacement of an olefin moiety with the ester group, and the quinoid sub-structure with
the triazole ring. The southern fragment needed for the macrocyclic ring formation was
obtained from Evans' syn aldol as the key reaction and with the use of D-mannitol as the
cheap source of a chiral starting material. For the synthesis of the northern fragment, we
utilized L-ascorbic acid, which provided the desired chiral functional groups at C6 and C7.
Further, the chain extension completed the synthesis of the northern fragment. In our
approach, the crucial 20 membered macrocyclic ring was formed employing the click
chemistry. When tested for their ability to directly trans-differentiate human mesenchymal
stem cells to neurons, two novel compounds (20a and 7) from this series were identified and
this was further validated by the presence of specific neuronal biomarkers (i.e. nestin, agrin
and RTN4).
2

Trans-differentiation of human mesenchymal stem cells to neurons by small molecules April 18, 2017
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Graphical Abstract
Day 2
Day 7
Day 8
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Trans-differentiation of human mesenchymal stem cells to neurons by small molecules April 18, 2017
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1. Introduction
Geldanamycin (Scheme 1, 1) is the first small molecule inhibitor of Heat Shock Protein 90
(Hsp90) and received a serious attention over the years.1-7 1-4,8-13 Hsp90 is known as a
molecular chaperone, and in this capacity, it plays a key role in protecting several misfolded
proteins, commonly known as client proteins.14-18 The key objectives of the molecular
chaperone are maintaining a proper folding of proteins, and in creating a balance between
protein synthesis and degradation. Through their binding to Hsp90, several natural products
and analogs, such as, herbimycin (2),19,20 also inhibit the chaperone process of various client
proteins and this then ultimately leads to protein degradation. Geldanamycin (1) binds to the
N-terminal domain of ATP binding site of Hsp90, and through this binding, it inhibits the
chaperone activity of a given client protein.3,21 This inhibition leads to the disruption of the
Hsp90-client protein complex. Following the subsequent ubiquitination, the unchaperoned
client proteins are then degraded by the proteasome. Due to the importance of several Hsp90
client proteins in signal transduction and the transcription machinery, geldanamycin and other
related natural products and their structural analogs have shown a great potential, not only in
cancer but several other drug discovery programs.9,22 The importance of geldanamycin
(1),12,17,23-25 and other natural products (for example: reblastatin, 3)26 that are known for
binding to Hsp90, led to several synthetic efforts in developing various efficient synthetic
methods and obtaining their analogs.10,13,27-35
O
O
OH
19-membered ring
MeO
MeO
O
O
O
1
1
1
N
N
N
H
H
H
O
O
MeO
MeO
MeO
OH
OH
OH
6
6
6
15
7
7
7
15
14
12
14
12
12
10
10
10
14
11
11
11
OCONH2 MeO
OCONH2
OCONH2
OMe
Geldanamycin, 1
OMe
Herbimycin, 2
OMe
Reblastatin, 3
Scheme 1: Geldanamycin (1) herbimycin, 2 and rablastatin, 3.
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2. Results and Discussion
2.1 Synthetic Planning
With the goal of developing a stereoselective synthesis for obtaining a diverse set of
macrocyclic compounds having a chiral key fragment, C10-C14, we were interested in
exploring the diversity of the north-western site. Our generic target is shown as 4 in Scheme
2. One of our objectives was developing a practical and scalable synthesis of fragment C10-
C14 which can further be utilized in obtaining three types of macrocyclic compounds, shown
as 5, 6 and 7. Our set targets have several attractive features, and these include: (i) 20-
membered macrocyclic ring formation using a click approach giving the triazole moiety, (ii)
connection of C10-C14 chiral sub-structure with C1-C7 chain through the lactone ring, and
(iii) the possibility of the reduced double at C4-C5. The replacement of the quinoid moiety
with the triazole may provide some additional binding contacts with the target protein.
Moreover, this could also be used in introducing a diversity site in our design strategy. To our
knowledge, a value to this approach in obtaining diverse macrocycles has not been explored
in the past. Shown in Scheme 3 is our key disconnection approach to the synthesis of
macrocyclic compounds, 5 and 6. In our plan, we envisioned two key fragments, i.e.
southern fragment, 8 and eastern fragment, 9, which following the connection, would serve as
the starting material to try our macrocyclic click reaction. Compound 8 can be obtained by a
chiral auxiliary-based syn aldol, giving synthon 10, which in turn, can be obtained from D-
mannitol, 11. We plan achieving the synthesis of an eastern fragment, 9 from L-ascorbic
acid, 13, a cheap chiral starting material via an allylic alcohol, 12.
Scheme 2: Geldanamycin-inspired compounds 5, 6 and 7.
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Scheme 3: Synthetic plan to obtain both fragments, 8 and 9.
2.2 Chemical Synthesis
Our synthesis of fragment 17 from D-mannitol (11) is shown in Scheme 4. In a simple three
step sequence, it was converted to 14 according to the literature procedure,36a which
following cyclization under acidic conditions, and the reduction of the double bond, gave the
corresponding lactone, 15.36b The asymmetric hydrogenation gave the relative 1,3-
stereochemistry which is also well-documented in the literature.36c This in a few simple
transformations allowed completing the synthesis of 17. Synthesis of the crucial southern
fragment with the key chiral auxiliary-based syn-aldol is shown in Scheme 5. Compound 18
obtained from 17 by oxidation was subjected to Evans' syn aldol36 ,37 reaction. This approach
d
produced the expected product as a major diastereomer, 20 (dr >98%). In a simple few steps,
20 was converted to 21 which upon removal of the chiral auxiliary, finally, gave the key
azido carboxylic acid, 22. Having a practical synthesis of the southern fragment 22 achieved,
we then focused on the eastern fragment 9. The synthesis from L-ascorbic acid (13) is
shown in Scheme 6. It was converted to 23 in three simple steps following the literature
procedure.38a This then led to producing the corresponding alcohol 24, which following
oxidation was then subjected to Wittig reaction using the literature method, 38b giving cis, 25
and trans, 26 as two inseparable isomers in 4:1 ratio (note - in both cases, the olefin geometry
was assigned on the basis of coupling constants; see experimental section). This mixture was
subjected to reduction, which allowed us separating the two geometrical isomers. In two
steps, 27 was obtained from the desired isomer. This was then subjected to an ester
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Trans-differentiation of human mesenchymal stem cells to neurons by small molecules April 18, 2017
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hydrolysis giving the free carboxylic acid which upon alkylation or amidation gave 28/29
respectively. Using 28/29, the required key eastern fragments, 30/31 (Scheme 6) were
produced.
Scheme 4: Synthesis of 17 from D-mannitol, 11.
southern fragment:
syn aldol
O
O
OH
OTBDPS
TiCl4, DIPEA
DCM, 2 h
O
OH
O
OTBDPS
12
14
N
12
10
14
O
18
+
11
N
O
OMe
82%, 2 steps
OMe
Bn
19
20
17
18
Bn
DMP, NaHCO3
DCM, 1 h, 80%
major diastereomer, de >99%
O
OH
O
1. PTSA, MeOH, 2 h
2. TsCl, TEA, DCM, 4 h
10
14
12
11
N
N3
O
3. NaN3, DMF, 2 h
65% 3 steps
OMe
21
Bn
O
1. Me3OBF4, PS
DCM, 1 h
MeO
10
12
11
14
OH
N3
2. H2O2, LiOH, 2 h
79%, 2 steps
OMe
22
Scheme 5: Our syn aldol approach to the synthesis of the southern fragment, 22 from 18.
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Trans-differentiation of human mesenchymal stem cells to neurons by small molecules April 18, 2017
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OH
HO
H
1. Acetone, CuSO4, 60 h
1. Ag2O, MeI
O
O
2. 30% H2O2, K2CO3, 24 h
OH
O
DMF, 1 h
HO
O
6
O
O
6
O
7
OEt
7
3. EtI, CH3CN, 70 oC, 24 h
80% 3 steps
2. LAH, THF, 12 h
83% 2 steps
HO
L-ascorbic acid, 13
OH
OMe
24
23
1. DMP, NaHCO3, DCM, 1 h
2. NaH, THF, -70 oC,
2 h, 69% 2 steps
inseparable (4:1) mixture
EtOOC
O
1. DIBAL-H, DCM, 80%, 1 h
2. DMP, NaHCO3, DCM, 2 h
O
O
6
trans
isomer
+
7
3. Ph3PCH(CH3)COOEt
Toluene, 80 oC, 4 h
64% 2 steps
PhO
O
P
COOEt
OMe
CO2Et
PhO
25
26
O
OMe
27
O
O
X
1
X
1. LiOH, THF
H2O, 16 h
1. HCl, THF, 4 h
O
OH
2. TIPSCl, Im
DCM, 1 h
O
2. Esterification
or
6
7
Amidation
OMe
, X = O, 76% 2 steps
29, X = NH, 62% 2 steps
TIPSO
, X = O, 84% 2 steps
31, X = NH, 86% 2 steps
OMe
28
30
Scheme 6: Completion of the eastern fragments synthesis
Our synthesis of geldanamycin-inspired macrocyclic compounds 5 and 6 that utilize click
39
chemistry38
as the key reaction is shown in Scheme 7. The precursors of the macrocyclic
c,
click, 32/33 were obtained from the coupling of 22 with 30/31. Upon subjection to CuI in the
presence of DIPEA under refluxing conditions, produced the macrocyclic products, 5 and 6.
In both cases, the formation of the macrocycle was confirmed by MS and NMR (note - a
separate singlet for one proton in the aromatic region from the 1,2,3-triazole ring; see the
experimental section).
Macrocyclic click to obtain 5 and 6:
X
N
N
X
1
O
O
N3
N
1. DCC, DMAP
DCM, 16 h
CuI, DIPEA
22
+
30/31
MeO
MeO
OMe O
OMe O
6
15
15
THF, reflux, 4 h
2. TBAF, THF
2 h
7
13
11
13
11
12
10
12
10
O
O
OMe
OH
OMe
OH
32
, X = O, 47% 2 steps
5, X = O,72%
33, X = NH, 52% 2 steps
6
, X = NH, 63%
Scheme 7: Completion of the synthesis of geldanamycin-inspired macrocyclic compounds, 5
and 6.
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In a similar manner, the synthesis for obtaining the dihydro-eastern fragment, 37 is shown in
Scheme 8. The synthesis details are provided in the supporting information. Compound 37
was then coupled with 22 giving the coupled product, 38, which upon the click reaction gave
the expected macrocyclic product, 7 (Scheme 9).
Scheme 8: Synthesis of the dihydro-eastern fragment, 37.
Scheme 9: Synthesis of geldanamycin-inspired macrocyclic compound, 7.
2.3 Molecular Docking Studies
Molecular docking simulations of 6 and 7 were performed with Hsp90 (see SI). The 3D-
structural coordinates of human Hsp90-geldanamycin complex were taken from the protein
data bank (PBD) with PDB id 1YET.21 In docking studies, the oxygen atoms of compound 6
are making hydrogen bonds with Asn51 and Lys112 residues of the binding site of protein.
Similarly, the hydroxyl group of 7 is making hydrogen bonds with Asn51 and Phe138
residues of the binding site. Additionally, the nitrogen atoms of the 1,2,3-triazole ring in
compound 7 are interacting with Lys58 via hydrogen bonding. A pi-cation interaction was
also observed between the triazole ring and Lys58 residue (see, Figure 1 and Table 1)
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Figure 1: Hsp90 ATP-binding site interactions with 6 and 7
GScore: Glide score; Lipophilic EvdW: Hydrophobic potential from the receptor at the ligand
atoms that are considered to be hydrophobic; H Bond: Rewards for hydrogen bonding
interaction between ligand and protein; Electro: Electrostatic reward; Expos Penal: Penalties
for exposed hydrophobic ligand part.
Table 1: Glide score and the contributing parameters (docking with Hsp90).
2.4 Trans-differentiation of Human Mesenchymal Stem Cells to Neurons by Small
Molecules
All the compounds obtained from this program were then evaluated for their ability to
directly trans-differentiate human stromal mesenchymal stem cells isolated from umbilical
cord tissue to neurons.40-47 48 Unlike the biological approaches47,49-51 that are commonly
utilized in promoting the differentiation of stem cells to neurons, we were interested in
41,46,53-55
examining the effect/transformation through small molecules.52-54
This offer
advantages, such as, (i) use of a well-defined single chemical entity that could be further
10

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optimized for the target specificity, (ii) ease of screening with clear phenotypic end points,
and (iii) novel therapeutic opportunities etc.
In a typical procedure, the umbilical cord tissue-derived mesenchymal stem cells from
healthy donors were cultured in DMEM complete medium supplemented with 10% FBS and
growth factors (see, Supporting Information). After 24 hrs, compounds were added to the
cellular screens at a final concentration of 10.0 µM. Forskolin and neuro-induction medium
(commercially available) were added as positive controls to a few wells. The phenotypic
changes in the cells were monitored by capturing any conspicuous visible changes in the
cellular morphology at regular intervals. Shown in Figure 2 are: (a) structures of two active
compounds 7 and 20a; (b and c) phenotypic changes towards extended neural processes (bi
and multi-polar) upon exposure to small molecules 7 and 20a; and, (d) the presence of
selected biomarkers (i.e. nestin, agrin and RTN4) identified in transformed neuronal cells, in
both cases.
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(a)
(b; shown with 7, 20X magnification)
(c; shown with 20a, 20X magnification)
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(d)
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Figure 2: (a) structures of two active compounds, (b and c) differentiation of stem cells to
neurons by 7 and 20a, and (d) identification of specific biomarkers for neuronal cells with 7
and 20a (note, NTC: no template control)
3.0 Experimental
The detailed experimental section is provided in the Supporting Information.
4.0 Conclusions
Thus, a new series of geldanamycin-inspired compounds was obtained that utilized a
macrocyclic click as the key step in the 20 membered ring formation. The diverse nature of
this approach can lead to several other related analogs. The computational studies indicated
better binding interactions with the ATP binding domain of Hsp90 protein with compound 7
compared to geldanamycin; although this need to be further validated by various biological
studies. Our interest in developing the chemical approaches for directly trans-differentiating
mesenchymal stem cells to neurons led the identification of two novel active compounds.
This was further supported by the identification of specific biomarkers (i.e. nestin, agrin and
RTN4). To gain a deeper mechanistic understanding of this finding, work is now being
extended for carrying-out genome-wide studies, and evaluating the scope of these compounds
as the neurogenesis or neuroprotective agents in specific neuro-disorders.
Acknowledgment
This work was supported by DST SERB grant. SJ thank CSIR India for the award of PhD
fellowship. Our sincere thanks to Dr. Ben Ross, The University of Queensland, Australia, for
assistance with molecular docking studies.
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