Derivatization of agelastatin A leading to bioactive analogs and a trifunctional probe

Article abstract of DOI:10.1016/j.bmcl.2016.02.051

(-)-Agelastatin A (AglA, 1), a member of the pyrrole-aminoimidazole marine alkaloid (PAI) family, possesses a unique tetracyclic structure and is one of the most potent anticancer PAIs isolated to date. In efforts to expand the SAR of these agents and del

Full text of DOI:10.1016/j.bmcl.2016.02.051

Accepted Manuscript
Derivatization of Agelastatin A Leading to Bioactive Analogs and a Trifunc-
tional Probe
Morgan Jouanneau, Brandon McClary, Jeremy C. Reyes, Rong Chen, Yuling
Chen, William Plunkett, Xin Cheng, Andrew Z. Milinichik, Earl F. Albone, Jun
O. Liu, Daniel Romo
PII:
S0960-894X(16)30163-9
DOI:
http://dx.doi.org/10.1016/j.bmcl.2016.02.051
BMCL 23605
Reference:
To appear in:
Bioorganic & Medicinal Chemistry Letters
Received Date:
Accepted Date:
29 January 2016
18 February 2016
Please cite this article as: Jouanneau, M., McClary, B., Reyes, J.C., Chen, R., Chen, Y., Plunkett, W., Cheng, X.,
Milinichik, A.Z., Albone, E.F., Liu, J.O., Romo, D., Derivatization of Agelastatin A Leading to Bioactive Analogs
and a Trifunctional Probe, Bioorganic & Medicinal Chemistry Letters (2016), doi: http://dx.doi.org/10.1016/j.bmcl.
2
016.02.051
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a
b
a§
c
c
c
Morgan Jouanneau , Brandon McClary , Jeremy C. Reyes , Rong Chen , Yuling Chen , William Plunkett , Xin
d
d
d
b
a,e *
Cheng , Andrew Z. Milinichik , Earl F. Albone , Jun O. Liu and Daniel Romo

Bioorganic & Medicinal Chemistry Letters
Derivatization of Agelastatin A Leading to Bioactive Analogs and a Trifunctional
Probe
a
c
b,§
d
d
Morgan Jouanneau, Brandon McClary, Jeremy C. Reyes, , Rong Chen, Yuling Chen, William
d
e
e
e
c
a,*
Plunkett, Xin Cheng, Andrew Z. Milinichik, Earl F. Albone, Jun O. Liu, and Daniel Romo
a
Department of Chemistry and Biochemistry, Baylor University, Waco, TX 76798, USA
b
Department of Chemistry, Texas A&M University, College Station, TX, 77842-3012.
c
Department of Pharmacology and Molecular Sciences, John Hopkins School of Medicine, 725 North Wolfe St., Baltimore, MD 21205, USA
d
Department of Experimental Therapeutics, The University of Texas MD Anderson Cancer Center, Houston, TX 77054, USA
e
Department of Biochemistry Discovery, Morphotek, Inc, 210 Welsh Pool Road, Exton, PA 19422, USA
§
Current address: The Dow Chemical Co, Freeport, TX.
Corresponding Author
*
ARTICLE INFO
ABSTRACT
Article history:
Received
Revised
Accepted
Available online
()-Agelastatin A (AglA, 1), a member of the pyrrole-aminoimidazole marine alkaloid (PAI)
family, possesses a unique tetracyclic structure and is one of the most potent anticancer PAIs
isolated to date. In efforts to expand the SAR of these agents and delineate sites that tolerate
modification while retaining activity, we synthesized several derivatives and tested their
anticancer activity. The cytotoxic effects of these derivatives were measured against several
cancer cell lines including cervical cancer (HeLa), epidermoid carcinoma (A431), ovarian (Igrov
and Ovcar3), osteosarcoma (SJSA1), acute T cell leukemia (A3), epidermoid carcinoma (A431)
in addition to primary human chronic lymphocytic leukemia cells. New positions for
modification of AglA and new substitutions were explored leading to novel derivatives, 14-
chloro AglA (3) and 14-methyl AglA (12), that retained activity toward various cancer cell lines
with decreased toxicity toward B- and T-cells. The SAR data informed the synthesis of a
trifunctional probe bearing an alkyne and a diazirine potentially useful for cellular target
identification.
Keywords:
Agelastatin A
Photoaffinity probe
Diazirine
Pyrrole-aminoimidazole alkaloid
Structure-activity relationship
2
009 Elsevier Ltd. All rights reserved.
(
)-Agelastatin A (1, AglA) is a tetracyclic member of the
providing a marked increase in potency toward a leukemia and a
breast cancer cell line, respectively. While AglA has been an
attractive target for both synthetic and biological studies over two
decades, a molecular level understanding of its effects in cells
including cellular target(s) has not been described. Herein, we
report expanded structure-activity relationship studies of
agelastatin A enabling the identification of an active chloro
derivative and the design and synthesis of a bioactive,
trifunctional cellular probe that could assist with cellular target
identification.
1
pyrrole-2-aminoimidazole alkaloid (PAI) family. Since its
isolation in 1993 by Pietra and co-workers from the sponge
AglAs dendromorpha, this family has grown to include six
agelastatin congeners (A through F, Figure 1a). Weinreb
described the first total synthesis of AglA and since then several
additional racemic and enantioselective syntheses of AglA have
been reported. Recently, Movassaghi developed an elegant and
efficient bioinspired approach to AglA and all congeners that
involved a late stage C4-C8 bond formation. Our group
2
3
4
5
6
7
6a
reported an alternative bioinspired approach that involved a final
8
N
pyrrole-C7 bond formation. The reported bioactivities of this
9
alkaloid are varied and include inhibition of -catenin,
suppression of osteopontin-mediated malignant transformation
and inhibition of glycogen synthase kinase (GSK-3b). AglA
and derivatives demonstrated good potency against leukemia1
and notably displayed excellent blood-brain barrier penetration.
1
0
11
2
These recent studies by Molinski and Yoshimitsu revealed that
substitutions at C13 on the pyrrole ring are tolerated (Figure 1c)
with addition of a C13-trifluoromethyl and chloro substituent

mono and dimethylated compounds 7, 8 was obtained in favor of
the dimethyl derivative. Stille conditions delivered only the
monomethyl derivative 7 in 77% yield. Hydrogenolysis of
bromide
7
gave 15-methyl-13,14-debromoAglA (9) in
quantitative yield (Scheme 2).
Figure 1. a) Agelastatin
A (1) and congeners. b) Structure-activity
relationship (SAR) overview of agelastatin A (1). c) Two known potent AglA
derivative
Scheme 2. Synthesis of methylated pyrrole AglA derivatives
Further substitutions on the pyrrole ring were investigated
including arylation, alkynylation and acylation (Scheme 3).
Suzuki coupling with phenylboronic acid gave a mixture of bis
and mono-arylation products providing 13,14-diphenyl derivative
Our derivatization of AglA focused on functionalization at
C14 and C15 of the pyrrole ring given the lack or absence of
investigations of these positions. Given the efficiency and
scalability of the Movassaghi synthesis of all known agelastatins,
we prepared ()-agelastatin A (1) for our studies as described
starting from D-aspartic acid in 9 steps in ~4% overall yield
10 and 14-phenyl derivative 11 derivatives in 23% and 19%
yields, respectively. Stille coupling with tetramethyltin and
tributylethynylphenyltin gave the 14-methyl derivative 12 and
14-ethynylphenyl derivative 14 in 26% and 55% yield,
respectively. Stille coupling with an enol ether stannane followed
by mild hydrolysis gave the C-acetylated derivative 13 in 46%
yield over two steps.
(
95% ee) and all derivatives prepared herein began with this
6a
material. Iodination with N-iodosuccinimide (NIS) promoted by
1
3
3
(InOTf)
gave 14-iodo 2 in 74% yield (Scheme 1) which
enabled subsequent functionalization of C14 through
chemoselective Pd-mediated reactions. Initial chlorination
conditions employing standard conditions (NCS) gave complex
®
14
mixtures, however chlorination of AglA with Palau’chlor gave
a mixture of the desired 14-chloro AglA 3 (24%) and AgelB 4
(30%) along with traces of C13,C14-dichlorinated derivative
detected by LCMS. Modified reaction conditions allowed access
®
to AgelB (4) in 5 minutes using KBr and selectfluor , as
15
brominating agent. Longer reaction times (1 h) with excess
brominating agent gave AgelB (4) with 15-bromoAgelB (5) in
46% yield, the first C15 functionalized AglA derivative. The 15-
iodoAgelB (6) was synthesized from AgelB (4) employing a
13
large excess of iodine in the presence of Lewis acid in 71%
yield over extended periods (2 days). Alternatively, treatment of
AglA with 1.5 equivalent of NBS followed by iodine at 60 °C for
16 h gave iodo derivative 6 in 60% yield.
Scheme 3. Synthesis of various C14 AglA derivatives 10-14
Modifications of the C5 and N9 positions were also studied
leading to several O- and N-substituted derivatives and a stable
aminal derivative. Triazole amide derivative 15 was obtained via
acid catalyzed C5 substitution with 4-pentyn-1-ol followed by
Sharpless-Hüisgen cycloaddition with -azido-N-benzylamide
(
A) to provide amide 15 in 46% (Scheme 4). Inspired by Pietra’s
16
work, acetylation of the C5 carbinolamine enabled milder
substitution conditions with nucleophiles such as 4-
hydroxybutanone to provide the alkyl carbinolamine 17 or
ammonia to give aminal 18 (Scheme 4). The presence of the
13
amine was confirmed by HRMS and by changes in the
C
chemical shift of C5; namely, a shift from 95.7 to 82.0 ppm
supports formation of an aminal at C5. This derivative exhibited
excellent stability in water. No trace of AglA by HPLC was
detected after 30 days in water at pH = 7 at 37 °C. To access the
unknown methyl-substituted N9 derivative 19, we took
advantage of the differential acidity of protons in AglA (C5-OH
Scheme 1. Synthesis of halogenated AglA derivatives 2-6
With various halogenated pyrrole derivatives in hand, we
studied methylation of the pyrrole ring to differentiate steric
versus electronic effects in this heterocycle beginning with C15.
Methylation at C15 was studied through both Stille and Suzuki
conditions (Scheme 2). Under Suzuki conditions, a mixture of the
>
N9-H > N3-H). Addition of 1.0 equiv of LiHMDS, presumably

led to initial deprotonation of the carbinolamine and addition of
TMSCl gave an intermediate silylether derivative that was not
isolated but directly treated with a second equivalent of LiHMDS
followed by quenching with methyliodide. The silylether was
then cleaved by direct addition of TBAF to provide the
monomethylated compound 19 in good yield (72%). The
regioselectivity of this methylation was confirmed through
HMBC correlations.
Table 1. Cytotoxicity of AglA Derivatives 1-14 and 25 with Pyrrole Ring
Me
Me
N
1
) Dowex 50W-X8
-pentyn-1-ol
23 C, 16 h
HO
N
5
i) LiHMDS,
TMSCl, MeI
DMF
HO
Modifications Toward CLL and HeLa Cell Lines.
O
N
N
Me
N
O
O
4
N
O
o
Br
H
H
Br
H
H
HN
Bn
3
O
NH
NH
Br
H
N
H
N
H
2) CuSO •5H2O
ii) TBAF
(72%)
H
NH
4
9NH
N
N
(Azide (A), THF/H O
Me
H
2
o
NH
50 C, 5 h
O
O
(46%, 2 steps)
1
O
15
19
Ac2O
py.
0
23 oC
16 h
O
Me
N
Me
Me
N
2
H N
O
H
O
N
5
O
AcO
NH3 (l), -30 oC
3 h
O
O
Br
H
Br
H
H
NH
OH
3 oC, 48 h
59%)
Br
H
NH
H
NH
N
N
H
H
2
N
then H2O
(54%)
H
NH
NH
(
NH
O
O
O
1
7
16
18
C5 = 95.7 ppm (AgelA)
C5 = 82.0 ppm
(C5 amino AgelA)
Scheme 4. Synthesis of O-substituted, N9 and aminal AglA derivatives 15,
7-19
1
Structure-activity relationship studies: anticancer
activity of AglA derivatives. AglA derivatives 2-25 were
screened against several cancer cell lines and primary chronic
lymphocytic leukemia (CLL) cells. HeLa cells were studied for
comparison to published cytotoxicity of previously described
AglA derivatives. Among the C14 and C15 AglA derivatives,
only 14-chloro AglA (3) and the 14-methyl derivative (12)
retained activity toward CLL albeit with only µM activity (IC50
=
2
1
.82 µM and 7.12 µM, respectively vs 0.71 µM for AglA, Table
). Likewise, toward HeLa cells, the choro derivative had greater
NT = Not tested.
activity than the methyl derivative (IC50 = 0.479 µM and 4.53
µM, respectively). Furthermore, the 14-methyl compound (12)
showed comparable potency when compared to AgelB (4) (HeLa,
IC50 = 4.53 µM and 7.85 µM, respectively). Given that a bromine
atom and a methyl group have similar steric size (VDW radii =
AglA derivatives 1, 3, 4 and 25 that displayed activity toward
CLL were also assayed against various cancer cell lines (Table 2,
CLL data included for comparison). The 14-chloro derivative 3
exhibited comparable activity to AglA while AgelB 4 was ~10X
less potent than AglA toward all cell lines. Notably, compound
1
7
1.85 and 1.80 Å, respectively) but different electronic
properties, this result suggests that the size of the group at C14 is
more important than electronic effects when considering activity
toward primary HeLa cells. This was further substantiated by
comparison to the C14-chloro derivative 3 for which the size
decreases from 1.85 Å (Br) to 1.75 Å (Cl) and the potency
significantly increased (17X toward HeLa cells). However,
introduction of electron-withdrawing groups at C14 such as
acetyl derivative (13) resulted in loss of activity (>10 µM and
2
5 showed comparable activity with parent natural product
against Igrov and Ovcar3 (IC50 0.306 and 0.276 µM,
respectively) whereas against SJSA1, A3 and A431 the IC50
values were above 1µM.
Table 2. Antitumor Activities of AglA Derivatives Toward Various Cancer
Cell Lines with Pyrrole Ring Modifications.
>
20 µM for CLL and HeLa, respectively, Table 1). While
previous reports demonstrated that a phenyl group is tolerated at
12
C13, for example toward triple-negative breast cancer cells, the
introduction of a phenyl (11) or ethynylphenyl (14) at C14 led to
loss of activity (>10 µM and >20 µM for CLL and HeLa,
respectively, Table 1). Similarly, the 13,14-diphenyl derivative
10 did not show significant activity against HeLa and CLL cell
lines. The 15-bromo and 15-methyl derivatives, 5 and 7, were
inactive against HeLa cells (Table 1). Additionally, no activity
was observed with the bis-methyl compound 8. These results
suggest that substitution at C15 is not tolerated for AglA
derivatives.
CLL (primary human cells), HeLa, Igrov (human ovarian), SJSA1
(osteosarcoma), A3 (human acute T cell leukemia), A431 (epidermoid
carcinoma) and Ovcar3 (ovarian carcinoma).
2b,11
Consistent with previously reported SAR studies,
O-
alkylated compounds 15, 17 and 20 suffered from loss of activity
toward HeLa and CLL cells (IC50 >10-20 µM, Table 3) with the
exception of derivative 17. However, we determined that AglA
was slowly regenerated from alkoxy carbinolamine 17 in water
(t1/2 = 16 h at 25 °C) likely from -elimination. This suggests that

this and related alkoxycarbinolamine derivatives of AglA could
be useful as prodrugs or alternatively the carbinolamine center
could serve as a point of attachment for antibody drug conjugate
synthesis if an appropriate pH sensitive alkoxycarbinolamine is
1
8
identified. On the other hand, the activity of aminal 18 was
significantly reduced toward HeLa cells (0.084 µM to 17 µM,
~
200X) and primary CLL cells (>30 µM) suggestive of the
stability of this aminal derivative (t1/2 = 7 days at pH = 5 at 37
C). Introduction of a N9-methyl group in derivative 19 resulted
°
in complete loss of activity (>20 µM against HeLa).
Figure 2. Effect of AglA (1) and 14-Cl-AglA (3) on B and T-cells and all
plasma cells from healthy donors.
Table 3. Cytotoxicity of AglA Derivatives 15-20 with Modifications at C5
and N9 Positions.
Synthesis of AglA Affinity Probes. Building on the SAR
gathered for the C14-methyl and chloro derivatives 3 and 12, we
targeted a photoaffinity probe that could be useful for affinity
1
9
chromatography experiments. Given the low potency exhibited
by such derivatives (~0.5-4.5 M) however, we elected to
synthesize a photoaffinity probe to improve the possibility of
capturing interacting proteins. Toward this goal, a trifunctional
probe 24 containing both a diazirine for covalent capture of
putative cellular target(s) through photocrosslinking and an
alkyne for subsequent biotin attachment to facilitate purification
of covalently modified proteins was synthesized (Scheme 5).
Beginning with previously prepared iodide 2, Stille coupling with
a stannyl bis-alkyne gave amine 21. Acylation of this amine with
diaziridine 22 gave amide 23 in modest yield with recovered
2
amine 21. Oxidation of the diaziridine with I in methanol
a
AglA (1) was released (t1/2 = 16 h at 25 °C in water); NT : not tested. Analog
9 is a racemate.
delivered the trifunctional diazirine probe 24. We were pleased to
find that both diaziridine 23 and diazirine 24 retained activity
toward HeLa cells albeit greatly reduced compared to AglA.
1
Protein binding studies. The propensity of AglA (1) and 14-Cl-
AglA (3) to bind to plasma proteins and their stability in these
media was investigated with both human plasma and fetal bovine
serum (FBS, Table 4). The cytotoxicity of the compounds did not
change dramatically when maintained in either human plasma or
FBS suggesting that they were not highly protein bound. Further
studies were performed to verify these results, and indeed full
recovery of these derivatives was possible also pointing to their
high stability. Low recovery is often an indication of material
loss during the analysis due to compound instability, non-specific
binding, or low solubility.
Table 4. Plasma Protein Properties of AglA (1) and 14-Cl-AglA (3)
Scheme 5. Synthesis of an AglA trifunctional probe 24
In summary, we expanded the SAR profile of the anticancer
agent, AglA through further investigations of the C14 position of
the pyrrole and positions not previously investigated, C15 and
N9. The 14-Cl-AglA (3) retained activity against various cancer
cell lines including HeLa, CLL and A431. This AglA derivative
also showed better selectivity toward CLL cells over B and T-
cells than AglA and exhibits low serum protein binding and good
stability given that recovery from serum was excellent. A
photoaffinity, trifunctional probe of AglA, albeit with low
cytotoxic activity relative to AglA, was synthesized and ongoing
photo-affinity chromatography studies will reveal the utility of
this probe. These studies, in conjunction with previous work by
others, demonstrate that few modifications of the AglA structure
are tolerated while retaining high potency relative to the parent
natural product. Finally, we demonstrated that alkoxyaminal
derivatives of AglA may prove useful as prodrugs given their
ability to regenerate AglA under mild hydrolysis conditions.
Studies toward cellular target identification, using this probe and
other strategies, in addition to further studies of potential
prodrugs are ongoing and will be disclosed in due course.
FBS: fetal bovine serum; Fraction bound (%): the portion of the compound
that is bound to human plasma; % recovery: the percentage of compound
recovered after the incubation period for plasma protein binding analysis.
Differential cytotoxicity studies. The toxicity of AglA (1)
and 14-Cl-AglA (3) was compared between CLL cells and
normal B and T-lymphocytes (Figure 2). The study revealed
that compared to CLL cells, AglA was less toxic toward B
and T cells from healthy donors. The selectivity for CLL
leukemia cells was even more apparent for 14-chloro
derivative 3. Although both compounds did demonstrate
toxicities toward normal B lymphocytes, this is generally not
a concern for future clinical application, as the B cell targeting
antibodies rituximab and ofatumumab were proven safe for
20
therapeutic treatment of B cell malignancies.
.

Acknowledgments
9.
Mason, C. K.; McFarlane, S.; Johnston, P. G.; Crowe, P.; Erwin,
P. J.; Domostoi, M. M.; Campbell, F. C.; Manaviazar, S.; Hale, K.
J.;Ei-Tanani, M. Mol. Cancer. Ther. 2008, 7, 548-558
We are grateful for financial support from NIH (R37
GM052964) to D.R., the Welch Foundation (AA-1280) to D.R.,
the Flight Attendant Medical Research Institute to J.O.L., and a
NRSA fellowship from NIH to B.M. Some of this work was
performed at Texas A&M University, Department of Chemistry.
Investigations using primary CLL cells were supported by the
CLL Global Research Foundation (IP13086447) to W.P. We
thank Dr. Howard Williams (Texas A&M University) for NMR
assistance and Dr. William G. Wierda (Univ. TX MD Anderson
Cancer Center) for providing the primary human CLL cells.
1
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Supplementary data including detailed experimental
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873.

Table 1
IC50 (µM)
HeLa
0.71 ± 0.10 0.084 ± 0.05
NT >20
2.82 ± 1.07 0.479 ± 0.31
Compound
X
Y
Z
CLL
AglA (1)
Br
Br
Br
Br
Br
Br
Br
H
H
I
H
H
2
3
Cl
Br
Br
Br
Me
H
H
AglB (4)
H
>30
>10
NT
7.85 ± 1.12
>20
5
7
8
9
Br
Me
Me
Me
H
>20
NT
>20
NT
>20
10
11
12
13
14
25
Ph
Br
Br
Br
Ph
Ph
Me
Ac
>10
>10
>20
H
>20
H
7.12 ± 1.44 4.53 ± 1.63
H
>10
>10
>20
>20
>20
Br PhCC
H
Me
H
H
9.77 ± 4.48
Table 2
IC50 (µM)
Cmpd
X
Y
H
CLL
HeLa
Igrov
SJSA1
A3
A431
Ovcar 3
AglA (1) Br
0.71 ± 0.10 0.084 ± 0.05 0.17 ± 0.00 0.44 ± 0.03 0.38 ± 0.05 0.46 ± 0.13 0.32 ± 0.09
3
Br Cl 2.82 ± 1.07 0.479 ± 0.31 0.26 ± 0.06 0.56 ± 0.02 0.47 ± 0.03 0.34 ± 0.01 0.54 ± 0.09
AglB (4) Br Br
Me
>30
7.85 ± 1.12
> 20
>1
>1
>1
>1
>1
>1
>1
>1
25
H
9.77 ± 4.48
0.31 ± 0.02
0.28 ± 0.00

Table 3
IC50 (µM)
cmpd
R
1
R
2
CLL
HeLa
AglA (1)
OH
H
0.71 ± 0.10 0.084 ± 0.05
15
H
>10
>20
a
a
a
1
7
H
H
5.74 ± 2.99
1.72 ± 0.53
17 ± 2.83
>20
1
1
2
8
9
0
NH
2
>30
OH
Me
H
NT
>10
>20
Table 4
IC50, µM
human plasma protein binding
fraction
cmpd
w/plasma
w/FBS
recovery (%)
bound (%)
AglA (1) 0.71 ± 0.10 0.93 ± 0.47
2.82 ± 1.86 2.65 ± 1.22
23.9
46.5
99.2
3
102.1