Symbiotic prodrugs (SymProDs) dual targeting of NFkappaB and CDK

Article abstract of DOI:10.1111/cbdd.13684

The release of an active drug from the prodrug generates a pro-fragment that typically has no biological activity and could result in adverse effects. By combining two drugs, wherein each drug acts as a pro-fragment of the other drug will eliminate the pr

Full text of DOI:10.1111/cbdd.13684

DR AMARNATH NATARAJAN (Orcid ID : 0000-0001-5067-0203)
Article type : Research Article
Symbiotic Prodrugs (SymProDs) Dual Targeting of NFkappaB and CDK
Sandeep Rana1, Smit Kour1, Yogesh A. Sonawane1, Caroline M. Robb1, Jacob I. Contreras1, Smitha
Kizhake1, Muhammad Zahid2, Adam R. Karpf1,5 and Amarnath Natarajan*,1,3,4,5
1Eppley Institute for Research in Cancer and Allied Diseases,
2Department of Environmental, Agricultural and Occupational Health,
3Department of pharmaceutical Sciences,
4Department of Genetics Cell Biology and Anatomy,
5Fred and Pamela Buffett Cancer Center
University of Nebraska Medical Center, Omaha, Nebraska 68198, USA
Running title:
SymProDs: A novel class of therapeutics
Keywords:
NFkappaB, CDK, Prodrugs
*Address for Correspondence:
Amarnath Natarajan PhD
Eppley Institute for Cancer Research and Allied Diseases
University of Nebraska Medical Center
This article has been accepted for publication and undergone full peer review but has not been
through the copyediting, typesetting, pagination and proofreading process, which may lead to
differences between this version and the Version of Record. Please cite this article as doi:
10.1111/CBDD.13684
This article is protected by copyright. All rights reserved

986805 Nebraska Medical Center, Omaha, NE 68198-6805, USA
TEL: (402) 559-3793
FAX: (402) 559 8270
Email: anatarajan@unmc.edu
Disclosure of Conflicts of Interest: Authors have no conflict of interest to disclose
This article is protected by copyright. All rights reserved

Abstract
The release of an active drug from the prodrug generates a pro-fragment that typically has no
biological activity and could result in adverse effects. By combining two drugs wherein each drug acts
as a pro-fragment of the other drug will eliminate the pro-fragment in the prodrug. Since they are
prodrugs of each other and are symbiotic we termed these as symbiotic prodrugs (SymProDs). To test
this idea, we generated SymProDs using NFB inhibitors that contain the reactive -methylene--
butyrolactone moiety and CDK inhibitors with solvent exposed secondary nitrogen atoms. We show
that secondary amine prodrugs of -methylene--butyrolactone containing NFB inhibitors undergo
slow release over a 72h period. Using an alkyne tagged secondary amine prodrug of -methylene--
butyrolactone containing NFB inhibitor, we demonstrate target engagement. The NFB-CDK
SymProDs were ~20-200 fold less active against the corresponding CDK inhibitors in in vitro CDK
kinase assays. Growth inhibition studies in a panel of ovarian cancer cell lines revealed potency trends
of the SymProDs mirrored those of the single treatments suggesting their dissociation in cells. In
conclusion our results suggest that SymProDs offer a productive path forward for advancing
compounds with reactive functionality and can be used as dual targeting agents.
This article is protected by copyright. All rights reserved

Introduction
An emerging concept is to incorporate prodrug design strategies early in the discovery phase
as opposed to a salvage effort when delivery strategies fail (Rautio, Meanwell, Di, & Hageman,
2018). By definition a prodrug inherently has far less to no pharmacological activity when compared
to the active drug but possess structural motifs that are liable to bioconversion that reveals the active
drug (Albert, 1958). The release of the active drug from the prodrug also generates a pro-fragment
that typically has no biological activity and, in some cases, could result in adverse effects. The
antibacterial field was the first to design “mutual prodrugs” also known as “codrugs” wherein two
drugs are conjugated through a labile linker (Baltzer et al., 1980). The marketed codrug sultamicillin,
upon hydrolysis, releases ampicillin (-lactamase antibiotic) and penicillanic acid sulfone (-
lactamase inhibitor). Bacteria develop resistance to -lactamase antibiotics by elevating the
expression of -lactamase, which is targeted by penicillanic acid sulfone in sultamicillin thus
restoring the efficacy of ampicillin.
Prodrug approaches to overcome high toxicity among chemotherapeutic agents have yielded
promising results in preclinical and clinical settings. Widely used drugs such as doxorubicin are
effective anticancer agents but suffer from offsite effects such as cardiomyopathy (Guthrie & Gibson,
1977). Aldoxorubicin (INNO-206) is a prodrug of doxorubicin that exploits the acidic tumor
microenvironment to trigger the release of doxorubicin (Kratz et al., 2002). The cardiotoxicity profile
of INNO-206 was far superior to doxorubicin in Phase II and III clinical trials (Cranmer, 2019).
Platinum based drugs are another class that is widely used to treat an array of cancers also suffers
from adverse effects. The reductive environment of the cancer cell allows the conversion of the
kinetically inert Pt(IV) low spin d6 octahedral complex to the square planar Pt(II) derivatives by
releasing the axial ligands (Hall, Mellor, Callaghan, & Hambley, 2007). The Gibson lab effectively
exploited this by incorporating bioactive axial ligands to trigger the release of cisplatin along with
COX and HDAC inhibitors in cancer cells (Babak et al., 2019; Harper et al., 2017; Petruzzella,
Braude, Aldrich-Wright, Gandin, & Gibson, 2017; Petruzzella, Sirota, Solazzo, Gandin, & Gibson,
2018).
An array of targeted anticancer agents contains enone (,-unsaturated carbonyl) moieties that
form covalent carbon-sulfur bonds with surface exposed sulfhydryl group of cysteine residues.
This article is protected by copyright. All rights reserved

Examples include FDA approved irreversible inhibitors ibrutinib and afatinib that target burton
tyrosine kinase and epidermal growth factor receptor, respectively (Pan et al., 2007; Solca et al.,
2012). Chemoproteomic studies suggest that cysteinome is susceptible to covalent kinase inhibitors
and since the off-targets also exhibit structure activity relationship a medicinal chemistry solution is
viable to achieve kinase selectivity (Lanning et al., 2014). Another class of anticancer agents are
pathway specific inhibitors that possess enone moieties which target the sulfhydryl groups of surface
exposed cysteine residues. In particular sesquiterpene natural products that contain an -methylene--
butyrolactone moiety that are known to form covalent adducts with proteins in the NFB pathway.
Parthenolide and isohelenin were early sesquiterpene lactones that were identified as NFB pathway
inhibitors (Bork, Schmitz, Kuhnt, Escher, & Heinrich, 1997). In an oral administered phase I trial,
parthenolide was not detected in the plasma suggesting stability issues (Curry et al., 2004). Secondary
amine-based prodrugs that masked the reactive enone of parthenolide improved stability while
maintaining anticancer activity in tumor models (Hexum et al., 2015; Ren, Yu, & Kinghorn, 2016).
Using a cell-based TNF-induced-IKK-mediated NFB luciferase reporter screen, we
identified an isatin-derived spirocyclic -methylene--butyrolactone (19) as an NFB inhibitor that
exhibited antitumor activity in an orthotopic model (Rana et al., 2016; Rana & Natarajan, 2013). Here
for the first time we show target engagement in cells by a secondary amine prodrug of an NFB
inhibitor with the -methylene--butyrolactone moiety. Kinetic studies suggest slow release of the
active enone. Several targeted agents contain secondary amines that are solvent exposed, for example
CDK inhibitor AT7519 has a piperidine ring and CDK4/6 inhibitor palbociclib has a piperazine ring.
Here we used the secondary amines of CDK inhibitors (AT7519 and palbociclib) to mask reactive -
methylene--butyrolactone containing NFB inhibitors (parthenolide and 19) to generate 4
compounds (5-8) that were prodrugs of each other (symbiotic prodrugs - SymProDs). Evaluation of
these SymProDs showed that the prodrug undergoes bioconversion to release the active enone in
cancer cells.
This article is protected by copyright. All rights reserved

Materials and Methods
All reagents were purchased from commercial sources and were used without further
purification. Flash chromatography was carried out on silica gel (200–400 mesh). Thin layer
chromatography (TLC) were run on pre-coated EMD silica gel 60 F254 plates and observed under
UV light at 254 nm and with basic potassium permanganate dip. Column chromatography was
1
performed with silica gel (230-400 mesh, grade 60, Fisher scientific, USA). H NMR (400 – 600
13
MHz) and C NMR (100 – 150 MHz) spectra were recorded in CDCl3, DMSO-d6 or CD3OD on a
1
13
Bruker instrument (CDCl3 was 7.26 ppm for H and 77.00 ppm for C, DMSO-d6 was 2.50 ppm for
13
1
13
1H and 39.00 ppm for C and CD3OD was 3.31 ppm for H and 49.3 for C. Proton and carbon
chemical shifts were reported in ppm relative to the signal from residual solvent proton and carbon.
Preparative HPLC was carried out on 250 x 21.2 mm C-18 column using gradient conditions (10 –
100% B, flow rate = 6.0 mL/min, 39 min). The eluents used were: solvent A (H2O with 0.1% Formic
acid) and solvent B (CH3CN with 0.1% Formic acid).
4-((4-(trifluoromethyl)piperidin-1-yl)methyl)-3,4-dihydro-5H-spiro[furan-2,3'-indoline]-2',5-dione
(19-AP)
4-(trifluoromethyl)piperidine (30 mg, 0.20 mmol) and TEA (50 L) were added sequentially
to a solution of 4-methylene-1'-(prop-2-yn-1-yl)-3,4-dihydro-5H-spiro[furan-2,3'-indoline]-2',5-dione
(21 mg, 0.10 mmol) in ethanol (2 mL) at RT. The resulting solution was stirred for 3 h. Then the
solvent was evaporated to afford the crude residue, which was purified by column chromatography to
afford the desired compound as a mixture of diastereomers. 1H NMR (DMSO-d6) δ 10.75 (d, J = 9.0
Hz, 1H), 7.56 (dd, J = 7.5, 28.3 Hz, 1H), 7.34 (q, J = 7.7 Hz, 1H), 7.06 (dt, J = 7.6, 14.2 Hz, 1H), 6.90
(d, J = 7.8 Hz, 1H), 3.05 – 2.62 (m, 5H), 2.44 – 1.91 (m, 5H), 1.77 (d, J = 12.6 Hz, 2H), 1.43 (tdd, J =
4.7, 8.7, 10.7, 12.7 Hz, 2H). 13C NMR (DMSO-d6) δ 177.98, 176.28, 142.94, 131.56, 126.90, 125.48,
123.06, 110.88, 81.57, 58.14, 52.85, 51.23, 38.39, 38.17, 36.37, 35.09, 24.80, 24.70. C18H19F3N2O3
[M]+: 368.14; found 369.75.
4-((dimethylamino)methyl)-3,4-dihydro-5H-spiro[furan-2,3'-indoline]-2',5-dione (19-AP2)
To a solution of 4-methylene-1'-(prop-2-yn-1-yl)-3,4-dihydro-5H-spiro[furan-2,3'-indoline]-
2',5-dione (60 mg, 0.28 mmol) in ethanol (3 mL) were added dimethylamine hydrochloride (45 mg,
0.56 mmol) and TEA (98 L). The resulting solution was stirred for 4 h and progress of the reaction
This article is protected by copyright. All rights reserved

was monitored by TLC. The solvent was evaporated to afford the crude residue, which was purified
1
by column chromatography to afford the desired compound as a mixture of diastereomers. H NMR
(CD3OD) δ 7.51 (d, J = 7.6 Hz, 1 Hz, 1H), 7.38 (t, J = 7.6 Hz, 1H), 7.13 (t, J = 7.6 Hz, 1H), 6.95 (d, J
= 7.6 Hz, 1H), 3.89 – 3.61 (m, 2H), 3.40 – 3.34 (m, 1H), 3.24 – 3.18 (m, 1H), 2.93 (s, 6H), 2.55 –
2.49 (m, 1H). 13C NMR (DMSO-d6) δ 177.88, 176.29, 142.36, 131.54, 126.91, 125.55, 123.05,
110.86, 81.58, 59.84, 45.59, 38.60, 36.36, C14H16N2O3 [M]+: 260.12; found 260.99.
1-(prop-2-yn-1-yl)indoline-2,3-dione (2)
To a solution of isatin (200 mg, 1.36 mmol) in anhydrous DMF (6 mL) cooled to 0°C under
argon atmosphere was added 60% NaH (82 mg, 2.04 mmol). After 10 min, propargyl bromide (0.19
mL, 2.04 mmol, 80%) was added at −0°C, and the solution was allowed to warm to ambient
temperature and stirred for 16h. Reaction mixture was washed with NH4Cl and extracted with EtOAc.
The organic phase was washed with brine, separated, dried over MgSO4, and the solvent was
evaporated in vacuo. The crude residue was purified by silica gel chromatography to afford the pure
product (192 mg, 75%). 1H NMR(CDCl3) δ 7.66 – 7.63 (m, 2H), 7.19 – 7.12 (m, 2H), 4.53 (d, J = 2.0
13
Hz, 2H), 2.30 (s, 1H). C NMR (CDCl3) δ 182.49, 157.13, 149.59, 138.39, 125.44, 124.18, 117.68,
111.06, 75.65, 73.31, 29.43. C11H7NO2 [M]+: 185.05; found 186.27.
Methyl 2-((3-hydroxy-2-oxo-1-(prop-2-yn-1-yl)indolin-3-yl)methyl)acrylate
In a round bottom flask, 1-(prop-2-yn-1-yl)indoline-2,3-dione (40 mg, 0.22 mmol) was
dissolved in THF: water (2 ml, 2:1) followed by addition of Indium powder (49 mg, 0.43 mmol) and
methyl 2-(bromomethyl) acrylate (77 mg, 0.43 mmol). Reaction was stirred at room temperature for
24h. Crude was dissolved in ethyl acetate and wash 1N HCl, and brine.
Crude mixture was column chromatographed using hexane and ethyl acetate gradient to obtain
1
methyl 2-((3-hydroxy-2-oxo-1-(prop-2-yn-1-yl)indolin-3-yl)methyl)acrylate (33 mg, 54% yield). H
NMR (CDCl3) δ 7.34 – 7.32 (m, 2H), 7.08 (t, J = 7.5 Hz, 1H), 7.03 (d, J = 8.0 Hz, 1H), 6.27 (s, 1H),
5.57 (s, 1H), 4.55 (dd, J = 2.5 Hz, 7.5 Hz, 1H), 4.35 (dd, J = 2.5 Hz, 7.5 Hz, 1H), 3.98 (br, 1H), 3.70
(s, 1H), 3.16 – 3.13 (m ,1 H), 2.76 – 2.73 (m, 1H), 2.24 – 2.23 (m, 1H). 13C NMR (CDCl3) δ 176.46,
168.23, 141.16, 134.06, 130.18, 129.67, 129.25, 124.60, 123.02, 109.42, 75.77, 72.48, 52.28, 40.23,
29.30. C16H15NO4 [M]+: 285.10; found 286.07.
4-methylene-1'-(prop-2-yn-1-yl)-3,4-dihydro-5H-spiro[furan-2,3'-indoline]-2',5-dione (3)
This article is protected by copyright. All rights reserved

Taken methyl 2-((3-hydroxy-2-oxo-1-(prop-2-yn-1-yl)indolin-3-yl)methyl)acrylate (33 mg, 0.12
mmol) in 3 mL of DCM and cooled to 0º C followed by addition of p-toluene sulfonic acid (45 mg,
0.24 mmol) Crude mixture was diluted in ethyl acetate and washed with brines, dried MgSO4, and
column chromatographed to yield 4-methylene-1'-(prop-2-yn-1-yl)-3,4-dihydro-5H-spiro[furan-2,3'-
indoline]-2',5-dione (18 mg, 61% yield). 1H NMR (CDCl3) δ 7.32 (dt, J = 7.5 Hz, 1 Hz, 1H), 7.34 (d,
J = 7.0 Hz, 1H), 7.16 (dt, J = 7.5 Hz, 0.5 Hz, 1H), 7.11 (d, J = 8.0 Hz, 1H), 6.42 (t, J = 2.5 Hz, 1H),
5.81 (t, J = 2.5 Hz, 1H), 4.59 – 4.38 (m, 2H), 4.35 (dd, J = 2.5 Hz, 7.5 Hz, 1H), 3.45 – 3.10 (m, 2H),
2.28 (t, J = 2.5 Hz, 1H). 13C NMR (CDCl3) δ 172.57, 168.77, 141.99, 132.52, 131.26, 126.53, 124.24,
124.03, 123.33, 110.04, 79.24, 75.99, 73.09, 36.32, 29.64.
1'-(prop-2-yn-1-yl)-4-((4-(trifluoromethyl)piperidin-1-yl)methyl)-3,4-dihydro-5H-spiro[furan-2,3'-
indoline]-2',5-dione (4)
4-(trifluoromethyl)piperidine (9 mL, 0.058 mmol) and TEA (20 mL) were added sequentially
to a solution of 4-methylene-1'-(prop-2-yn-1-yl)-3,4-dihydro-5H-spiro[furan-2,3'-indoline]-2',5-dione
(11 mg, 0.038 mmol) in methanol (1 mL) at RT. The resulting solution was stirred for 4 h. Then the
solvent was evaporated to afford the crude residue, which was purified by column chromatography to
afford the desired compound as a mixture of diastereomers. 1H NMR (CD3OD) δ 7.58 (d, J = 7.6 Hz,
1H), 7.53 – 7.49 (m, 1H), 7.26 – 7.21 (m, 2H), 4.57 – 4.55 (m, 2H), 3.98 – 3.75 (m, 3H), 3.50 – 3.45
(m, 1H), 3.17 – 3.13 (m, 2H), 2.96 – 2.91 (m, 1H), 2.78 (t, J = 2.4 Hz, 1H), 2.69 – 2.56 (m, 3H), 2.23
– 2.20 (m, 2H), 1.94 – 1.88 (m, 2H). UPLC−MS calculated for C21H21F3N2O3 [M]+: 406.41; found
406.79.
4-((4-(6-((6-Acetyl-8-cyclopentyl-5-methyl-7-oxo-7,8-dihydropyrido[2,3-d]pyrimidin-2-
yl)amino)pyridin-3-yl)piperazin-1-yl)methyl)-3,4-dihydro-5H-spiro[furan-2,3'-indoline]-2',5-dione
(5)
To a solution of 4-methylene-3,4-dihydro-5H-spiro[furan-2,3'-indoline]-2',5-dione (20 mg, 0.093
mmol), triethylamine in methanol (2 mL) was added palbociclib (42 mg, 0.093 mmol) and the mixture
was stirred for 48 h. Then the mixture was concentrated in vacuo and purified by prep HPLC (Yield
10 mg, 16%). 1H NMR (DMSO-d6) δ 10.24 (s, 1H), 10.08 (d, J = 3.5 Hz, 1H), 8.95 (s, 1H), 8.04 (dd,
J = 3.1, 12.2 Hz, 1H), 7.84 (dd, J = 2.6, 9.0 Hz, 1H), 7.66 – 7.40 (m, 1H), 7.27 – 7.02 (m, 2H), 7.02 –
6.87 (m, 1H), 6.77 (dd, J = 7.7, 1H), 6.02 (d, J = 11.6 Hz, 1H), 5.95 (d, J = 1.6 Hz, 1H), 5.82 (d, J =
This article is protected by copyright. All rights reserved

8.8 Hz, 1H), 5.46 (d, J = 1.5 Hz, 1H), 3.52 (s, 1H), 3.44 (s, 1H), 3.16 (d, J = 5.3 Hz, 2H), 3.08 (s, 1H),
2.94 (d, 1H), 2.89 – 2.52 (m, 3H), 2.44 (t, J = 5.6 Hz, 1H), 2.42 (s, 2H), 2.43 – 2.10 (m, 5H), 1.99 –
1.85 (m, 2H), 1.80 – 1.71 (m, 1H), 1.62 – 1.53 (m, 1H), 0.93 (t, J = 7.1 Hz, 3H). 13C NMR (DMSO-
d6) δ 202.90, 178.87, 167.23, 155.21, 142.02, 135.11, 130.98, 129.47, 128.40, 125.20, 121.55, 109.92,
99.98, 76.00, 51.98, 40.60, 40.43, 40.27, 40.10, 39.93, 39.41, 38.81, 31.78, 28.02, 25.57, 14.09
UPLC−MS calculated for C36H38N8O5 [M]+: 662.29; found 663.30.
6-Acetyl-8-cyclopentyl-2-((5-(4-(((3R,3aS,9aR,10aS,10bS,E)-6,9a-dimethyl-2-oxo-
2,3,3a,4,5,8,9,9a,10a,10b-decahydrooxireno[2',3':9,10]cyclodeca[1,2-b]furan-3-yl)methyl)piperazin-
1-yl)pyridin-2-yl)amino)-5-methylpyrido[2,3-d]pyrimidin-7(8H)-one (6)
To a solution of parthenolide (25 mg, 0.100 mmol), triethylamine in methanol (2 mL) was
added palbociclib (45 mg, 0.100 mmol) and the mixture was stirred for 48 h. Then the mixture was
concentrated in vacuo and purified by prep HPLC (Yield 16 mg, 23%). 1H NMR (DMSO-d6) δ 10.08
(d, 1H), 8.95 (s, 1H), 8.05 (dd, J = 3.0, 1H), 7.84 (t, J = 9.7 Hz, 1H), 7.51 – 7.41 (m, 1H), 5.86 – 5.78
(m, 1H), 3.98 (t, J = 9.1 Hz, 1H), 3.17 (d, J = 5.1 Hz, 4H), 3.06 (t, J = 4.8 Hz, 1H), 2.89 – 2.78 (m,
2H), 2.65 (dd, J = 7.6, 2H), 2.42 (s, 3H), 2.31 (s, 4H), 2.19 (d, 4H), 2.02 (t, 2H), 1.82 (d, 6H), 1.61 (d,
6H), 1.20 (s, 3H). 13C NMR (DMSO-d6) δ 175.82, 134.70, 125.12, 81.84, 68,28, 65.79, 61.59, 59.14,
48.24, 45.22, 40.99, 40,61, 40.27, 40.10, 39.93, 39.59, 36.50, 29.08, 24.18, 17.33, 17.01UPLC−MS
calculated for C39H49N7O5 [M]+: 695.38; found 696.38.
4-(2,6-dichlorobenzamido)-N-(1-((2',5-dioxo-4,5-dihydro-3H-spiro[furan-2,3'-indolin]-4-
yl)methyl)piperidin-4-yl)-1H-pyrazole-3-carboxamide (7)
To a solution of 4-methylene-3,4-dihydro-5H-spiro[furan-2,3'-indoline]-2',5-dione (16 mg,
0.074 mmol), triethylamine in methanol (2 mL) was added AT7519 (31 mg, 0.074 mmol) and the
mixture was stirred for 48 h. Then the mixture was concentrated in vacuo and purified by prep HPLC
1
(Yield 8 mg, 18%). H NMR (DMSO-d6) δ 10.76 (d, 1H), 10.27 (d, 1H), 10.16 (d, J = 8.6 Hz, 1H),
8.43 – 8.22 (m, 2H), 7.70 – 7.42 (m, 3H), 7.28 – 6.87 (m, 3H), 6.86 – 6.69 (m, 1H), 3.58 – 3.43 (m,
2H), 3.24 (s, 2H), 2.94 – 2.61 (m, 2H), 2.43 – 2.05 (m, 2H), 2.04 – 1.83 (m, 2H), 1.80 – 1.37 (m, 4H).
13C NMR (DMSO-d6) δ 178.50, 173.77, 163.12, 159.87, 141.15, 134.86, 131.40, 130.85, 130.76,
130.62, 128.53, 127.94, 124.52, 123.97, 123.34, 122.14, 120.97, 120.93, 109.12, 74.04, 60.14, 51.82,
This article is protected by copyright. All rights reserved

51.49, 50.64, 45.54, 37.91, 37.77, 30.53. LC−MS calculated for C28H26Cl2N6O5 [M]+: 596.13; found
597.2.
4-(2,6-dichlorobenzamido)-N-(1-(((3R,3aS,9aR,10aS,10bS,E)-6,9a-dimethyl-2-oxo-
2,3,3a,4,5,8,9,9a,10a,10b-decahydrooxireno[2',3':9,10]cyclodeca[1,2-b]furan-3-yl)methyl)piperidin-
4-yl)-1H-pyrazole-3-carboxamide (8)
To a solution of parthenolide (16 mg, 0.064 mmol), triethylamine (20 µL) in methanol (2 mL) was
added AT7519 (30 mg, 0.070 mmol) and the mixture was stirred for 48 h. Then the mixture was
concentrated in vacuo and purified by prep HPLC (Yield 10 mg, 25%). 1H NMR (DMSO-d6) δ 13.39
(s, 1H), 10.17 (s, 1H), 8.37 – 8.28 (m, 2H), 7.61 – 7.49 (m, 3H), 5.25 – 5.17 (m, 1H), 3.95 (t, J = 9.1
Hz, 1H), 3.72 (m, 1H), 2.84 (d, J = 11.5 Hz, 1H), 2.76 (d, J = 9.1 Hz, 2H), 2.70 – 2.53 (m, 3H), 2.54
(s, 2H), 2.35 (m, 1H), 2.26 – 2.06 (m, 2H), 2.06 – 1.92 (m, 3H), 1.66 – 1.54 (m,, 5H), 1.20 (s, 3H),
13
1.12 (td, J = 5.8, 12.7 Hz, 1H). C NMR (DMSO-d6) δ 177.33, 163.07, 160.72, 135.82, 135.08,
133.39, 132.34, 131.70, 128.88, 124.60, 121.92, 121.10, 82.05, 66.14, 61.55, 56.86, 53.88, 52.74,
47.99, 46.41, 45.62, 41.09, 36.62, 31.88, 31.59, 29.48, 24.08, 17.30, 17.2 LC-MS calculated for
C31H37Cl2N5O5 [M]+: 629.2; found 630.3.
Cell lines and Materials: The cell lines were assembled by Dr. Karpf and the original source is in
parenthesis. Cell line identity was confirmed by STR testing and were confirmed to be mycoplasma
free. Typical experiments were conducted with cells passaged at least 3-times following a thaw.
A2780 (ATCC), OVSAHO (Japanese collection of research bioresources cell bank) and SNU119
(Korean cell line bank) cells were maintained in RPMI medium (HyClone #SH30027.01) and
OVCAR4 (NCI division of cancer treatment and diagnosis cell line repository), OVCAR5 (Dr. Mitra,
Indiana University), OVCAR8 (NCI division of cancer treatment and diagnosis cell line repository),
Caov3 (Dr. Mitra, Indiana University), FT282-E1 (Dr. Drapkin – PMCID: PMC4517944) and FT282-
C11 (Dr. Karpf, clonal isolate – PMCID: PMC6244393) cells were maintained in DMEM high
glucose medium (HyClone #SH30022.01). All cell lines were supplemented with 10% FBS (Gibco
#26140-079) and 1% Penicillin-Streptomycin (HyClone # SV30010), cultured at 5% CO2, 37°C.
Immunoblotting: A2780 cells were treated with 20 μM of alkyne tagged 19-AP2 (analog 4) for 48h.
Lysates were prepared with lysis buffer consisting of 50 mM Tris (pH 7.5), 100 mM NaCl, 2 mM
EDTA, 1% NP-40, 0.1% SDS, 1mM PMSF, 1X protease and phosphatase inhibitors cocktails. Lysed
This article is protected by copyright. All rights reserved

cell extracts were incubated for 30 min on ice and vortexed every 15 min. Lysates were centrifuged at
14,000 g for 10 min at 4°C. Finally, supernatant was collected, and protein was quantified using BCA
protein assay kit (Pierce #23225). 2mg/mL (360 L) of protein was subjected to click chemistry.
Biotin-TAMRA-Azide (20 μL, 10 mM stock solution in DMSO); tris(2-carboxyethyl)phosphine
hydrochloride (TCEP) (10 μL, 100 mM freshly prepared stock solution in water); tris[(1-benzyl-1-H-
1,2,3-triazol-4-yl)methyl]amine (TBTA) (20 μL, 13.5 mM stock solution in 3:1 tertbutanol:DMSO);
CuSO4•5H2O (10 μL, 100 mM freshly prepared stock solution in water) for a total reaction volume of
60 μL and added 580 μL of PBS buffer (1X) to make total volume of 1 mL. Samples were incubated
for 3 hours at the room temperature and 15 μL of samples was stored at -20 C as control. The
samples were then separated on a monomeric avidin column according to the manufacturer’s
instructions (Pierce) at 4 °C. After the samples were collected, they were concentrated using a 10 kDa
molecular weight cutoff filter (Amicon), ddH O (50 mL) was added, centrifuged, and finally the
2
samples were freeze-dried. Protein samples were run on 4-15% gradient SDS-PAGE gels (BioRad) in
1x Tris- Glycine-SDS buffer at 120V for 60min. Proteins were transferred onto PVDF membranes by
using semi-dry transfer method. Membranes were blocked with 5% fat-free milk in 1x Tris bufferd
saline with 0.1% Tween (TBST) for 1h at RT. Membranes were incubated overnight at 4°C with
primary antibodies against IKK (CST #8943), p65 (SantaCruz #sc372), IB (CST #4812), IKK
(Millipore #05-631) and -Tubulin (CST #3873) made in 5% fat-free milk in 1x TBST. Membranes
were incubated with the appropriate HRP-conjugated secondary antibody (Thermo Fisher Scientific
#G-21040 and #32460) for 1h at RT. All membranes washes were performed using 1x TBST buffer
(20 mM Tris-HCl (pH 7.5), 150 mM NaCl, 0.1% Tween-20) 3 times for 10 min. ECL Prime (GE
Healthcare #RPB2236) was used to develop blots.
Growth inhibition assay: Cells were seeded at density of 4000 cells/well in a 96 well plate in RPMI
and allowed to adhere overnight at 5% CO2, 37°C. On the following day, cells were treated with
different concentrations of analog 19, analog 19-AP1 and analog 19-AP2. PrestoBlue reagent
(Invitrogen) was added according to manufacturer’s instruction to cells after 72 h drug treatment to
assess the growth inhibition. Fluorescence excitation/ emission was measured at 560/590 nm using
plate reader SpectraMax M5e instrument. IC50 was determined using SigmaPlot (Systat Software, San
Jose, CA).
This article is protected by copyright. All rights reserved

Cysteine Reactivity Assay: 200mM tris base buffer was diluted in 1X methanol (pH ~ 8.8). L-
cysteine reduced was diluted to a final concentration of 250 μM in assay buffer (100 mM Tris pH 8.8,
50% methanol as a co-solvent). In triplicate, 100 μL of L-cysteine (250 μM) in assay buffer was
added to each well in a clear, flat-bottom 96-well plate (Costar, Corning). Analog 19 (2.5 L, 10 mM
stock, 250 M final), analog 19-AP2 (2.5 L, 10 mM stock, 250 M final) and DMSO were added to
each well in triplicate (10 min interval) and incubated at room temperature for 0-60 min. Upon
incubation completion, Ellman’s reagent (5 μL, 100 mM stock in DMSO, final concentration 5 mM)
was added and the reaction incubated at room temperature for 10 minutes. Absorbance was measured
at 412 nM on a plate reader (SpectramaxM5e).
Glutathione Assay: Standard preparation – From the 1 mg GSH Standard (10 µg/ µL) provided, add
H2O to make a working std (2.5 mg/mL). Make dilutions with sample buffer provided to give 11
standards of 100 µL each ranging from 0.5 mg/ml of GSH up to 0.0005 mg/mL of GSH. Standard 12
is blank. Standards were prepared in duplicates.
Cell lysate preparation – 1 × 106 Ovarian cancer cells (A2780 and OVSAHO) rinsed 3X with ice-cold
1X PBS, were harvested and centrifuged at 14,000 rpm for 15 minutes at 4°C to pellet the cells. 100
µL of the lysis buffer from the abcam kit (ab 65322) was added to the tubes and incubated on ice for
10 min. Centrifuge at 14,000 rpm for 10 minutes at 4°C to pellet the debris. 75 µL supernatant was
carefully removed and transferred to an ice-cold tube. 25 µL 4M PCA was added to the supernatant to
precipitate the proteins. The solution was vortexed and incubated on ice for 5 min, centrifuged at
14,000 rpm for 5 minutes at 4°C to pellet the precipitate. 88 µL supernatant was carefully transferred
to another chilled tube. 30 µL ice cold 2 M KOH was added to this supernatant and mixed well to
neutralize the PCA. pH was adjusted to 6.5 – 8 range with 0.01M KOH. Proteins were pelleted at
14,000 rpm for 15 minutes and 100 µL supernatant was transferred to another chilled tube.
Sample preparation – 40 µL of the lysate was diluted to 100 µL with buffer. 6 half dilutions were
made for each cell lysate in duplicates.
Detection reagent – 1:1 volume of GST reagent and MCB (monochlorobimane) were mixed to create
a master mix. 4 µL of this mix was added per well.
This article is protected by copyright. All rights reserved

Detection – The assay was done in a 96 well black, clear bottom plate and the final reaction volume
was 104 µL. The plate was incubated at 37°C and read on the Spectramax in the kinetic mode every 2
minutes for an hour. Flourescence was measured at Ex/Em 380/461.
Data processing – Data was analyzed and a time point was picked where the curves were flat
indicating a stable GSH/GSSG-MCB adduct formation. The time point used was 28 minutes.
Standard graphs were plotted for both high and low concentrations and only the low concentration
demonstrated linearity. All values were blank subtracted to obtained corrected readings.
Concentration of GSH (mg/mL) was calculated for the highest concentration in each cell line from the
standard graph. Dilution factor (from PCA and KOH addition) was accounted for and the corrected
blank subtracted values were obtained. The final values were expressed as GSH concentration in µM
/106 cells.
19F NMR Experiment: A solution of glutathione (55 mg) in water (3.58 mL) and DMSO (8.35 mL)
was titrated to pH 7.4 using 1.0 M NaOH (water). Next, glutathione solution (2.7 mL, pH 7.4) was
treated with a 300 L (10 mM stock solution of compound 19-AP2 in DMSO). An aliquot (500L of
19
19-AP2 and 50 L DMSO-d6) was transferred to NMR tube for F NMR at 0, 24, 48 and 72h at 80
C.
This article is protected by copyright. All rights reserved

Results and Discussion
Reaction of 4-methylene-3,4-dihydro-5H-spiro(furan-2,3'-indoline)-2',5-dione (19)(Rana et
al., 2016; Rana & Natarajan, 2013) with 4-trifluoropiperidine in ethanol resulted in the corresponding
amino prodrugs 4-((4-(trifluoromethyl)piperidin-1-yl)methyl)-3,4-dihydro-5H-spiro(furan-2,3'-
indoline)-2',5-dione (19-AP) as a mixture of diastereomers (Figure 1A). The -methylene--
butyrolactone moiety on analog 19 is reactive toward biological nucleophiles such as glutathione and
cysteine. To determine if secondary amine prodrug of 19 (19-AP) results in improved stability, we
compared the reactivity of analog 19 and the prodrug 19-AP in a cysteine binding assay using
Ellman’s reagent (Figure 1B).(Zaro, Whitby, Lum, & Cravatt, 2016) Analog 19 reacted rapidly with
free cysteine whereas amine-prodrug 19-AP is more stable under similar conditions. These studies
show that the amine-based prodrugs of -methylene--butyrolactone containing compounds are stable
to biological nucleophiles. Next, to assess the kinetics of the release of the active compound 19 from
the prodrug 19-AP, we monitored the release of 4-trifluoropiperidine from the prodrug 19-AP using
19F-NMR spectroscopy following the method reported by the Colby group.(Woods, Mo, Bieberich,
19
Alavanja, & Colby, 2011) The F NMR studies showed a time-dependent disappearance of the CF3
signal on 19-AP and corresponding appearance of the CF3 signal on 4-trifluoromethylpiperidine
(Figure 1C). This observation is consistent with previously reported studies with a prodrug of
parthenolide. These studies clearly show that 19-AP is more stable to biological nucleophiles and the
prodrug is hydrolyzed slowly to release the active compound 19 over a 72h period.
We next explored whether prodrug 19-AP covalently binds to NFB pathway proteins in cells.
Scheme 1 summarizes the synthesis of an alkyne-tagged prodrug (4) that is suitable for in situ click
chemistry. Briefly, commercially available isatin (1) was stirred with propargyl bromide and
potassium carbonate in DMF to yield substituted isatin (2). Intermediate 2 was subjected to Indium
catalyzed Barbier-type reaction followed by a acid-catalyzed cyclization to yield compound 3.(Rana
& Natarajan, 2013) Treatment of 3 with 4-trifluoromethylpiperidine and triethylamine in methanol
resulted in desired alkyne-tagged analog 4.
To determine target engagement in cells, we subjected cancer cells (A2780) with the alkyne-
tagged prodrug 4 for 48h. Cells were then harvested, washed and pelleted. Unbound 4 was removed
by extensively washing the cell pellet. The cell pellets were lysed, and 2 mg/mL of lysates were
This article is protected by copyright. All rights reserved

subjected to click chemistry with TAMRA-Biotin-Azide trifunctional probe (10 mM stock, 20 L,
click chemistry tools), and other click reagents (TCEP, 100 mM stock, 20 L; TBTA 13.5 mM stock,
20 L and CuSO4 100 mM stock, 10 L) to make total volume of 1 mL. The reaction was incubated
for 3h at RT. The lysates were incubated with monomeric avidin beads for additional 1h. The sample
was washed with 15 mL elution buffer to remove unbound proteins followed by multiple washing
with regeneration buffer (15 mL) to cleave biotin-monomeric avidin bond (Pierce® Monomeric
Avidin Kit, ThermoScientific, Cat # 20227). The collected regeneration sample was washed with an
excess of water (50 mL) to remove salts and freeze dried to yield tagged protein powder (Note: Even
after multiple washing, proteins samples contained significant amounts of salts). Protein powder was
dissolved in buffer and subjected to Western blot analyses. The membranes were probed for NFB
pathway proteins IKK, RELA, IB and IKK (Figure 2). We observed covalent binding of IKK,
and RELA proteins to analog 4 but not IB or IKK proteins despite presence of surface exposed
cysteine residues. This is the first report that shows that an amine-based prodrug of -methylene--
butyrolactone containing NFB inhibitor selectively engaged NFB pathway proteins RELA and
IKK in cells. This confirms the release of the parent eneone (3) from prodrug 4, which irreversibly
binds to RELA and IKK.
We next evaluated if the amine-prodrugs (19-AP and 19-AP2) inhibited cancer cell growth.
Analogs 19, 19-AP and 19-AP2 were screened in ovarian cancer lines (A2780, and OVSAHO) and
immortalized fallopian tube epithelial cells (FT282E1) in a 72h growth inhibition assay (Figure 3). In
A2780 and FT282E1 cell lines, amine-prodrugs are ~2  3 fold less active than the parent compound
19 whereas amine-prodrugs 19-AP and 19-AP2 are more potent in the OVSAHO cell line (Figure 3).
To determine if this difference in activities was due to quenching of the eneone by the thiol
nucleophile glutathione (GSH) or release kinetics, we determined cellular GSH levels in the ovarian
cancer cell lines (Supplementary Figure S1). We did not observe any correlation between the GSH
levels and the activity (Figure 3D). The growth inhibitory activity observed with 19-AP and 19-AP2
suggests that the active eneones are released because others and we have previously reported that
reduction of the exocyclic double bond in compounds with -methylene--butyrolactone resulted in a
complete loss of activity.(Guzman et al., 2007; Holcomb et al., 2012; Nasim & Crooks, 2008;
This article is protected by copyright. All rights reserved

Neelakantan, Nasim, Guzman, Jordan, & Crooks, 2009; Pei et al., 2016; Rana et al., 2016;
Shanmugam et al., 2011; Shanmugam et al., 2010)
Two plausible mechanisms for the conversion of amine-prodrugs to parent analogs have been
previously suggested.(Holcomb et al., 2012; Shanmugam et al., 2011; Shanmugam et al., 2010)
Figure 3E summarizes the plausible mechanisms for the conversion of 19-AP to analog 19. In the first
case, GSH functions as a nucleophile to drive the E2 elimination of the protonated piperidine nitrogen
of 19-AP to generate 19 (Figure 3E, I-1). In the second case, the secondary amine in 19-AP is
oxidized facilitating a Cope like elimination to generate 19 (Figure 3E, I-2). Since it is known that the
GSH levels are elevated in cancer cell lines (Balendiran, Dabur, & Fraser, 2004) the former
mechanism is generally accepted for the release of the active drug. However, in our hands, we did not
see a correlation between the GSH levels and growth inhibition (Figure 3D). This suggests that analog
19 is probably generated from 19-AP through the later mechanism.
Our studies show that secondary amine-based prodrugs generated from 19 engages RELA and
IKK in cells and inhibits cancer cell growth. Therefore, we hypothesized that prodrugs of 19
generated with secondary amine containing inhibitors that target a complimentary pathway will allow
simultaneous targeting of two pathways. To test this hypothesis, we synthesized two prodrugs each of
analog 19 (5 and 7) and parthenolide (6 and 8) with CDK4/6 inhibitor palbociclib (5 and 6), which
has been approved for the treatment of metastatic breast cancer patients and AT7519 (7 and 8), which
is a pan-CDK inhibitor currently in clinical trials (Figure 4A).
To evaluate the change in vitro efficacy of the palbociclib hybrids (5 and 6) for CDK4 and
CDK6, we performed cell-free assays (Figure 4B). Although the CDK6:palbociclib co-crystal
structure (pdb id: 5L21) suggests that the piperazine nitrogen atom is solvent exposed, palbociclib
hybrids with 19 and parthenolide (hybrid 5 and 6) were less active than palbociclib against CDK4 and
CDK6. The 19-palbociclib hybrid (5) was ~100- and ~20-fold less potent than palbociclib against
CDK4 and CDK6 respectively. The parthenolide-palbociclib hybrid (6) was ~200- and ~50-fold less
potent than palbociclib against CDK4 and CDK6 respectively. It is clear that CDK6 tolerates
alkylation of piperazine nitrogen atom better than CDK4. We observed a similar effect during the
development of a CDK6 selective degrader using PROTAC based strategy.(Rana et al., 2019)
Moreover, the larger size of parthenolide compared to analog 19 is probably responsible for a greater
This article is protected by copyright. All rights reserved

loss of activity of the parthenolide-hybrid (6) compared to analog 19-hybrid (5). Nevertheless, it is
clear that blocking the piperazine nitrogen on palbociclib results in significant loss of activity, which
will be regained in the cells upon the release of the NFB inhibitors (analog 19 and parthenolide).
Therefore, we termed these hybrids as symbiotic prodrugs (SymProDs).
CDK4, CDK5, and CDK6 show a negative Pearson and Spearman correlation with RELA
each with a p value < 0.05 (depmap.org) suggesting that simultaneous targeting of these CDKs and
RELA would result in synergistic effects. To test this idea, we evaluated the (palbociclib, AT7519,
19, parthenolide) four SymProDs (5-8) and their corresponding 1:1 mixture in growth inhibition
assays in a panel of five cell lines (Figure 5). Among the inhibitors treated as single agents the
potency irrespective of the cell line ranked as follows: AT7519 > 19 > parthenolide > palbociclib. The
1:1 combination treatment showed dose-response across all cell lines, and the potency mirrored the
dominant single agent treatment. Surprisingly, SymProDs were less potent than the corresponding 1:1
combinational treatment. As expected, the AT7519 based SymProDs were more potent than the
palbociclib based SymProDs. The potency trends of the hybrids followed those of the single
treatments which suggests that the SymProDs are dissociating in the cells. The average combination
index (AveCI) at effective dose (ED) 50, 75 and 90 for SymProD 5 in OVSAHO cells was 0.9
indicating weak synergism while the AveCI > 1.0 for the FT282E1 cells.
Although significant advancements have been made to treat cancer, ovarian cancer remains
the leading cause of gynecological cancer deaths in the United States. Several reports show that NFB
pathway is dysregulated in ovarian cancer. (House et al., 2017; Lee et al., 2007; Leizer et al., 2011;
Sagher et al., 2014; White et al., 2011) In a panel of ovarian cancer cell lines, we observe elevated
levels of RELA when compared to immortalized fallopian tube epithelial cell lines, which are an
appropriate normal control for most ovarian cancers(Perets & Drapkin, 2016) (Supplementary Figure
S2). Consistently, analyses of the TCGA data from 17 cancer types show that RELA (p65) expression
is the highest in ovarian tumors (Supplementary Figure S3). These suggest that targeting the NFB
pathway proteins (RELA and IKK) is a viable therapeutic strategy for ovarian cancer. Moreover,
CDK4/6 has been implicated in ovarian cancer tumorigenesis and resistance to therapy.(Dall'Acqua et
al., 2017; Gao et al., 2017) Therefore, we wanted to compare the efficacy of the palbociclib based
SymProDs (5 and 6) with the corresponding (1:1) mixtures in and expanded panel of 9 ovarian cancer
This article is protected by copyright. All rights reserved

(CaOV3, OVCAR5, OVCAR8, A2780, Kuramochi, OVCAR4, OVSAHO, SKOV3 and SNU119) and
2 immortalized fallopian tube epithelial cell lines (FT282E1 and FT282C11) (Figure 6).
Analyses of the data show that on an average SymProD 6 was ~7-fold less potent than the
corresponding 1:1 mixture, while the 19-based SymProD 5 was equipotent to the corresponding 1:1
mixture. The AveCI for SymProD 5 in SKOV3 cells was 0.2 indicating strong synergism. Although the
parthenolide:palbociclib (1:1) mixture was on an average ~2-fold more potent than the 19:palbociclib
(1:1) mixture, the 19-palbociclib SymProD 5 was ~3-fold more potent than the parthenolide-
palbociclib SymProD 6. This could be attributed to lower potency of parthenolide-palbociclib
SymProD 6 in the in vitro kinase assays. An alternate possibility is that the release of palbociclib from
19 is more efficient than the release of palbociclib from parthenolide. These results support the
development of SymProDs with -methylene--butyrolactone moiety containing NFB inhibitors and
known drugs that have secondary amine functionality as a means to target two pathways
simultaneously. A similar idea was recently reported by Neumann et al., where in INVDA led to the
symbiotic formation of two active prodrugs.(Neumann, Gambardella, Lilienkampf, & Bradley, 2018)
Conclusion
In conclusion, we synthesized two amine-based prodrugs of analog 19, which was previously
reported as an NFB pathway inhibitor. The prodrugs were more stable to biological nucleophiles,
and the associated slow release was characterized by 19F NMR studies. Using an alkyne-tagged
prodrug (4), we show for the first-time engagement of NFB pathway proteins by an analog with -
methylene--butyrolactone moiety in cells. The prodrugs exhibited anticancer effects in growth
inhibition assays. Using CDK inhibitors that contain solvent exposed secondary amines and NFB
inhibitors 19 and parthenolide, we synthesized 4 SymProDs. Screening these SymProDs in cell lines
revealed that 19 derived SymProDs were more potent than parthenolide derived SymProDs both in
cell free and cell-based assays. SymProDs are a viable path forward for advancing reactive
compounds with -methylene--butyrolactone moiety as NFB inhibitors. Additional mechanistic
and in vivo efficacy studies with SymProDs are currently underway and will be reported in due
course.
Acknowledgements
This article is protected by copyright. All rights reserved

This work was supported in part by NIH grants CA182820, CA197999 and CA036727. SKo and JIC
were supported by UNMC fellowship. We would like to thank the Natarajan lab members for helpful
discussions.
This article is protected by copyright. All rights reserved

Data Availability Statement
The data that support the findings of this study are available from the corresponding author
upon request.
References:
Albert, A. (1958). Chemical aspects of selective toxicity. Nature, 182(4633), 421-422. Retrieved from
https://www.ncbi.nlm.nih.gov/pubmed/13577867
Babak, M. V. V., Zhi, Y., Czarny, B., Toh, T. B., Ooi, L., Chow, E. K., . . . Pastorin, G. (2019). Dual-
targeting Dual-action Platinum(IV) Platform for Enhanced Anticancer Activity and Reduced
Nephrotoxicity. Angew Chem Int Ed Engl. doi:10.1002/anie.201903112
Balendiran, G. K., Dabur, R., & Fraser, D. (2004). The role of glutathione in cancer. Cell Biochem
Funct, 22(6), 343-352. doi:10.1002/cbf.1149
Baltzer, B., Binderup, E., von Daehne, W., Godtfredsen, W. O., Hansen, K., Nielsen, B., . . . Vangedal,
S. (1980). Mutual pro-drugs of beta-lactam antibiotics and beta-lactamase inhibitors. J
Antibiot
(Tokyo),
33(10),
1183-1192.
Retrieved
from
https://www.ncbi.nlm.nih.gov/pubmed/6969724
Bork, P. M., Schmitz, M. L., Kuhnt, M., Escher, C., & Heinrich, M. (1997). Sesquiterpene lactone
containing Mexican Indian medicinal plants and pure sesquiterpene lactones as potent
inhibitors of transcription factor NF-kappaB. FEBS Lett, 402(1), 85-90. Retrieved from
https://www.ncbi.nlm.nih.gov/pubmed/9013864
Cranmer, L. D. (2019). Spotlight on aldoxorubicin (INNO-206) and its potential in the treatment of
soft tissue sarcomas: evidence to date. Onco Targets Ther, 12, 2047-2062.
doi:10.2147/OTT.S145539
Curry, E. A., 3rd, Murry, D. J., Yoder, C., Fife, K., Armstrong, V., Nakshatri, H., . . . Sweeney, C. J.
(2004). Phase I dose escalation trial of feverfew with standardized doses of parthenolide in
patients
with
cancer.
Invest
New
Drugs,
22(3),
299-305.
doi:10.1023/B:DRUG.0000026256.38560.be
This article is protected by copyright. All rights reserved

Dall'Acqua, A., Sonego, M., Pellizzari, I., Pellarin, I., Canzonieri, V., D'Andrea, S., . . . Baldassarre, G.
(2017). CDK6 protects epithelial ovarian cancer from platinum-induced death via FOXO3
regulation. EMBO Mol Med, 9(10), 1415-1433. doi:10.15252/emmm.201607012
Gao, Y., Shen, J., Choy, E., Mankin, H., Hornicek, F., & Duan, Z. (2017). Inhibition of CDK4 sensitizes
multidrug resistant ovarian cancer cells to paclitaxel by increasing apoptosiss. Cell Oncol
(Dordr), 40(3), 209-218. doi:10.1007/s13402-017-0316-x
Guthrie, D., & Gibson, A. L. (1977). Doxorubicin cardiotoxicity: possible role of digoxin in its
prevention.
Br
Med
J,
2(6100),
1447-1449.
Retrieved
from
https://www.ncbi.nlm.nih.gov/pubmed/589261
Guzman, M. L., Rossi, R. M., Neelakantan, S., Li, X., Corbett, C. A., Hassane, D. C., . . . Jordan, C. T.
(2007). An orally bioavailable parthenolide analog selectively eradicates acute myelogenous
leukemia stem and progenitor cells. Blood, 110(13), 4427-4435. Retrieved from
http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?cmd=Retrieve&db=PubMed&dopt=Citation
&list_uids=17804695
Hall, M. D., Mellor, H. R., Callaghan, R., & Hambley, T. W. (2007). Basis for design and development
of platinum(IV) anticancer complexes.
doi:10.1021/jm070280u
J
Med Chem, 50(15), 3403-3411.
Harper, B. W. J., Petruzzella, E., Sirota, R., Faccioli, F. F., Aldrich-Wright, J. R., Gandin, V., & Gibson, D.
(2017). Synthesis, characterization and in vitro and in vivo anticancer activity of Pt(iv)
derivatives of [Pt(1S,2S-DACH)(5,6-dimethyl-1,10-phenanthroline)]. Dalton Trans, 46(21),
7005-7019. doi:10.1039/c7dt01054k
Hexum, J. K., Becker, C. M., Kempema, A. M., Ohlfest, J. R., Largaespada, D. A., & Harki, D. A. (2015).
Parthenolide prodrug LC-1 slows growth of intracranial glioma. Bioorg Med Chem Lett,
25(12), 2493-2495. doi:10.1016/j.bmcl.2015.04.058
Holcomb, B. K., Yip-Schneider, M. T., Waters, J. A., Beane, J. D., Crooks, P. A., & Schmidt, C. M.
(2012). Dimethylamino parthenolide enhances the inhibitory effects of gemcitabine in human
pancreatic cancer cells. J Gastrointest Surg, 16(7), 1333-1340. doi:10.1007/s11605-012-1913-
7
This article is protected by copyright. All rights reserved

House, C. D., Jordan, E., Hernandez, L., Ozaki, M., James, J. M., Kim, M., . . . Annunziata, C. M. (2017).
NFkappaB Promotes Ovarian Tumorigenesis via Classical Pathways That Support Proliferative
Cancer Cells and Alternative Pathways That Support ALDH(+) Cancer Stem-like Cells. Cancer
Res, 77(24), 6927-6940. doi:10.1158/0008-5472.CAN-17-0366
Kratz, F., Warnecke, A., Scheuermann, K., Stockmar, C., Schwab, J., Lazar, P., . . . Unger, C. (2002).
Probing the cysteine-34 position of endogenous serum albumin with thiol-binding
doxorubicin derivatives. Improved efficacy of an acid-sensitive doxorubicin derivative with
specific albumin-binding properties compared to that of the parent compound. J Med Chem,
45(25), 5523-5533. Retrieved from https://www.ncbi.nlm.nih.gov/pubmed/12459020
Lanning, B. R., Whitby, L. R., Dix, M. M., Douhan, J., Gilbert, A. M., Hett, E. C., . . . Cravatt, B. F.
(2014). A road map to evaluate the proteome-wide selectivity of covalent kinase inhibitors.
Nat Chem Biol, 10(9), 760-767. doi:10.1038/nchembio.1582
Lee, D. F., Kuo, H. P., Chen, C. T., Hsu, J. M., Chou, C. K., Wei, Y., . . . Hung, M. C. (2007). IKK beta
suppression of TSC1 links inflammation and tumor angiogenesis via the mTOR pathway. Cell,
130(3),
440-455.
Retrieved
from
http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?cmd=Retrieve&db=PubMed&dopt=Citation
&list_uids=17693255
Leizer, A. L., Alvero, A. B., Fu, H. H., Holmberg, J. C., Cheng, Y. C., Silasi, D. A., . . . Mor, G. (2011).
Regulation of inflammation by the NF-kappaB pathway in ovarian cancer stem cells. Am J
Reprod Immunol, 65(4), 438-447. doi:10.1111/j.1600-0897.2010.00914.x
Nasim, S., & Crooks, P. A. (2008). Antileukemic activity of aminoparthenolide analogs. Bioorg Med
Chem Lett, 18(14), 3870-3873. doi:10.1016/j.bmcl.2008.06.050
Neelakantan, S., Nasim, S., Guzman, M. L., Jordan, C. T., & Crooks, P. A. (2009). Aminoparthenolides
as novel anti-leukemic agents: Discovery of the NF-kappaB inhibitor, DMAPT (LC-1). Bioorg
Med Chem Lett, 19(15), 4346-4349. doi:10.1016/j.bmcl.2009.05.092
Neumann, K., Gambardella, A., Lilienkampf, A., & Bradley, M. (2018). Tetrazine-mediated
bioorthogonal
prodrug-prodrug
activation.
Chem
Sci,
9(36),
7198-7203.
doi:10.1039/c8sc02610f
This article is protected by copyright. All rights reserved

Pan, Z., Scheerens, H., Li, S. J., Schultz, B. E., Sprengeler, P. A., Burrill, L. C., . . . Palmer, J. T. (2007).
Discovery of selective irreversible inhibitors for Bruton's tyrosine kinase. ChemMedChem,
2(1), 58-61. doi:10.1002/cmdc.200600221
Pei, S., Minhajuddin, M., D'Alessandro, A., Nemkov, T., Stevens, B. M., Adane, B., . . . Jordan, C. T.
(2016). Rational design of a parthenolide-based drug regimen that selectively eradicates
acute myelogenous leukemia stem cells.
doi:10.1074/jbc.A116.750653
J
Biol Chem, 291(48), 25280.
Perets, R., & Drapkin, R. (2016). It's Totally Tubular....Riding The New Wave of Ovarian Cancer
Research. Cancer Res, 76(1), 10-17. doi:10.1158/0008-5472.CAN-15-1382
Petruzzella, E., Braude, J. P., Aldrich-Wright, J. R., Gandin, V., & Gibson, D. (2017). A Quadruple-
Action Platinum(IV) Prodrug with Anticancer Activity Against KRAS Mutated Cancer Cell Lines.
Angew Chem Int Ed Engl, 56(38), 11539-11544. doi:10.1002/anie.201706739
Petruzzella, E., Sirota, R., Solazzo, I., Gandin, V., & Gibson, D. (2018). Triple action Pt(iv) derivatives of
cisplatin: a new class of potent anticancer agents that overcome resistance. Chem Sci, 9(18),
4299-4307. doi:10.1039/c8sc00428e
Rana, S., Bendjennat, M., Kour, S., King, H. M., Kizhake, S., Zahid, M., & Natarajan, A. (2019).
Selective degradation of CDK6 by a palbociclib based PROTAC. Bioorg Med Chem Lett.
doi:10.1016/j.bmcl.2019.03.035
Rana, S., Blowers, E. C., Tebbe, C., Contreras, J. I., Radhakrishnan, P., Kizhake, S., . . . Natarajan, A.
(2016). Isatin Derived Spirocyclic Analogues with alpha-Methylene-gamma-butyrolactone as
Anticancer Agents: A Structure-Activity Relationship Study. J Med Chem, 59(10), 5121-5127.
doi:10.1021/acs.jmedchem.6b00400
Rana, S., & Natarajan, A. (2013). Face selective reduction of the exocyclic double bond in isatin
derived spirocyclic lactones. Org Biomol Chem, 11(2), 244-247. doi:10.1039/c2ob27008k
Rautio, J., Meanwell, N. A., Di, L., & Hageman, M. J. (2018). The expanding role of prodrugs in
contemporary drug design and development. Nat Rev Drug Discov, 17(8), 559-587.
doi:10.1038/nrd.2018.46
This article is protected by copyright. All rights reserved

Ren, Y., Yu, J., & Kinghorn, A. D. (2016). Development of Anticancer Agents from Plant-Derived
Sesquiterpene Lactones. Curr Med Chem, 23(23), 2397-2420. Retrieved from
https://www.ncbi.nlm.nih.gov/pubmed/27160533
Sagher, E., Hernandez, L., Heywood, C., Pauly, G. T., Young, M. R., Schneider, J., . . . Annunziata, C. M.
(2014). The small molecule NSC676914A is cytotoxic and differentially affects NFkappaB
signaling in ovarian cancer cells and HEK293 cells. Cancer Cell Int, 14, 75.
doi:10.1186/s12935-014-0075-y
Shanmugam, R., Kusumanchi, P., Appaiah, H., Cheng, L., Crooks, P., Neelakantan, S., . . . Sweeney, C.
J. (2011). A water soluble parthenolide analog suppresses in vivo tumor growth of two
tobacco-associated cancers, lung and bladder cancer, by targeting NF-kappaB and generating
reactive oxygen species. Int J Cancer, 128(10), 2481-2494. doi:10.1002/ijc.25587
Shanmugam, R., Kusumanchi, P., Cheng, L., Crooks, P., Neelakantan, S., Matthews, W., . . . Sweeney,
C. J. (2010). A water-soluble parthenolide analogue suppresses in vivo prostate cancer
growth by targeting NFkappaB and generating reactive oxygen species. Prostate, 70(10),
1074-1086. doi:10.1002/pros.21141
Solca, F., Dahl, G., Zoephel, A., Bader, G., Sanderson, M., Klein, C., . . . Adolf, G. R. (2012). Target
binding properties and cellular activity of afatinib (BIBW 2992), an irreversible ErbB family
blocker. J Pharmacol Exp Ther, 343(2), 342-350. doi:10.1124/jpet.112.197756
White, K. L., Rider, D. N., Kalli, K. R., Knutson, K. L., Jarvik, G. P., & Goode, E. L. (2011). Genomics of
the NF-kappaB signaling pathway: hypothesized role in ovarian cancer. Cancer Causes
Control, 22(5), 785-801. doi:10.1007/s10552-011-9745-4
Woods, J. R., Mo, H., Bieberich, A. A., Alavanja, T., & Colby, D. A. (2011). Fluorinated amino-
derivatives of the sesquiterpene lactone, parthenolide, as (19)f NMR probes in deuterium-
free environments. J Med Chem, 54(22), 7934-7941. doi:10.1021/jm201114t
Zaro, B. W., Whitby, L. R., Lum, K. M., & Cravatt, B. F. (2016). Metabolically Labile Fumarate Esters
Impart Kinetic Selectivity to Irreversible Inhibitors. J Am Chem Soc, 138(49), 15841-15844.
doi:10.1021/jacs.6b10589
This article is protected by copyright. All rights reserved

Figure Legends
Figure 1:
Stability and kinetics of release studies with a prodrug (19-AP1) of the NFB
inhibitor (19). (A) Structures of the NFB inhibitor 19 and its amino prodrug 19-AP1, and 19AP2.
(B) Cysteine reactivity assay used to compare the relative reactivity of the 19-AP1 and 19 towards
cysteine. (C) 19F NMR study to monitor the kinetics of the conversion of 19-AP1 to 19 as a function
of free 4-trifluoromethylpiperidine.
Figure 2:
Synthesis of an alkyne-tagged 19-AP1 (4) and cellular target engagement studies.
(A) Synthetic scheme that was used for the generation of the alkyne-tagged 19-AP1 analog (4). (B)
Cancer cells (A2780) were incubated with 10M of analog 4 for 24h. In the cells analog 4 will be
converted to analog 3 with an active methylene group which will react with surface exposed cysteine
residues on proteins. The above lysate was subjected to click reagents (TCEP, TBTA and CuSO4) and
azido-biotin. Biotin-tagged-3-bound proteins were subjected to mono-avidin column. Biotin-tagged
proteins were captured, the column was washed to removed untagged proteins and the biotin-tagged-
3-bound proteins were eluted with regeneration buffer (6M urea/PBS). The eluted lysates were
subjected to Western blot analyses and probed for proteins (IKK, RELA, IB and IKK) in the
IKK complex.
Figure 3:
Growth inhibition assays with NFB inhibitor and its prodrugs. (A-C) Dose
response studies with analog 19, 19-AP1 and 19-AP2 in ovarian cancer cell line A2780, immortalized
fallopian tube epithelial cells FT282E1 and ovarian cancer cell line OVSAHO. (D) Table shows the
IC50 values derived from curve fitting the growth inhibition data and the glutathione levels in A2780,
FT282E1 and OVSAHO cell lines. (E) Plausible mechanism for the conversion of the prodrug to the
active drug.
Figure 4:
Symbiotic Prodrugs (SymProDs) of NFB and CDK inhibitors. (A) Structure of the
symbiotic prodrugs generated from CKD4/6 inhibitor palbociclib, CDK2/5/9 inhibitor AT7519 and
This article is protected by copyright. All rights reserved

NFB inhibitors 19 and parthenolide. The CDK inhibitors are shown in blue. SymProd 5 (palbociclib
+ 19), SymProd 6 (palbociclib + parthenolide), SymProd 7 (AT7519 + 19) and SymProd 8 (AT7519 +
parthenolide). (B) Evaluation of palbociclib, SymProDs 5 and 6 in CDK4 and CDK6 in vitro kinase
assays
Figure 5:
SymProDs screened for growth inhibitory activity. Summary of growth inhibition
assays in FTE282E1, OVSAHO, HPNE, MiaPaCa2 and S2013 cell lines with palbociclib, AT7519,
19, parthenolide, (1:1) combinations and the corresponding SymProDs (5-8) (n= 3, bar graph is Mean
SD)
±
Figure 6:
Growth inhibition assays in a panel of Ovarian cell lines. Growth inhibition assays
in a panel of 11 cell lines (9 ovarian cancer cell lines and 2 immortalized fallopian tube epithelial cell
lines) with SymProDs 5 and 6 and their corresponding 1:1 mixture. (n = 3)
This article is protected by copyright. All rights reserved

Figure 1
This article is protected by copyright. All rights reserved

Figure 2
This article is protected by copyright. All rights reserved

Figure 3
This article is protected by copyright. All rights reserved

Figure 4
This article is protected by copyright. All rights reserved