Design, synthesis, and pharmacological evaluation of thapsigargin analogues for targeting apoptosis to prostatic cancer cells

Article abstract of DOI:10.1021/jm010985a

A series of thapsigargin (TG) analogues, containing an amino acid applicable for conjugation to a peptide specifically cleaved by prostate-specific antigen (PSA), has been prepared to develop the drug-moiety of prodrugs for treatment of prostatic cancer. The analogues were synthesized by converting TG into O-8-debutanoylthapsigargin (DBTG) and esterifying O-8 of DBTG with various amino acid linkers. The compounds were evaluated for their ability to elevate the cytosolic Ca²⁺ concentration ([Ca²⁺]_i) in TSU-Pr1 cells, their ability to inhibit the rabbit skeletal muscle SERCA pump, and their ability to induce apoptosis in TSU-Pr1 human prostatic cancer cells. The activity of analogues, in which DBTG were esterified with ω-amino acids [HOOC(CH₂)_nNH₂, n = 5-7, 10, 11], increased with the linker length. Analogues with 3-[4-(L-leucinoylamino)phenyl]propanoyl, 6-(L-leucinoylamino)hexanoyl, and 12-(L -serinoylamino)dodecanoyl were considerably less active than TG, and analogues with 12-(L-alaninoylamino)dodecanoyl and 12-(L-phenylalaninoylamino)dodecanoyl were almost as active as TG. The 12-(L-leucinoylamino)dodecanoyl gave an analogue equipotent with TG, making this compound promising as the drug-moiety of a PSA sensitive prodrug of TG.

Full text of DOI:10.1021/jm010985a

4696
J . Med. Chem. 2001, 44, 4696-4703
Design , Syn th esis, a n d P h a r m a cologica l Eva lu a tion of Th a p siga r gin An a logu es
for Ta r getin g Ap op tosis to P r osta tic Ca n cer Cells
Carsten M. J akobsen,† Samuel R. Denmeade,‡ J ohn T. Isaacs,‡ Alyssa Gady,‡ Carl E. Olsen,§ and
Søren Brøgger Christensen*,†
Department of Medicinal Chemistry, The Royal Danish School of Pharmacy, 2 Universitetsparken,
DK-2100 Copenhagen, Denmark, Department of Experimental Therapeutics, J ohns Hopkins Oncology Center,
Bunting-Blaustein Cancer Research Building, 1650 Orleans Street, Baltimore, Maryland 21231, USA, and Chemistry
Department, The Royal Veterinary and Agricultural University, 17 Bu¨lowsvej, DK-1870 Frederiksberg, Denmark
Received J uly 13, 2001
A series of thapsigargin (TG) analogues, containing an amino acid applicable for conjugation
to a peptide specifically cleaved by prostate-specific antigen (PSA), has been prepared to develop
the drug-moiety of prodrugs for treatment of prostatic cancer. The analogues were synthesized
by converting TG into O-8-debutanoylthapsigargin (DBTG) and esterifying O-8 of DBTG with
various amino acid linkers. The compounds were evaluated for their ability to elevate the
cytosolic Ca2+ concentration ([Ca2+]i) in TSU-Pr1 cells, their ability to inhibit the rabbit skeletal
muscle SERCA pump, and their ability to induce apoptosis in TSU-Pr1 human prostatic cancer
cells. The activity of analogues, in which DBTG were esterified with ω-amino acids [HOOC-
(CH2)nNH2, n ) 5-7, 10, 11], increased with the linker length. Analogues with 3-[4-(L-leucinoyl-
amino)phenyl]propanoyl, 6-(L-leucinoylamino)hexanoyl, and 12-(L-serinoylamino)dodecanoyl
were considerably less active than TG, and analogues with 12-(L-alaninoylamino)dodecanoyl
and 12-(L-phenylalaninoylamino)dodecanoyl were almost as active as TG. The 12-(L-leucinoyl-
amino)dodecanoyl gave an analogue equipotent with TG, making this compound promising as
the drug-moiety of a PSA sensitive prodrug of TG.
Ch a r t 1a
In tr od u ction
Thapsigargin (TG, 1) (Chart 1) is a sesquiterpene-γ-
lactone isolated from seeds and roots of the umbelli-
ferous plant, Thapsia garganica L.1,2 TG selectively
inhibits the ubiquitous sarcoplasmic and endoplasmic
reticulum Ca2+-dependent ATPases (SERCA’s) with an
apparent dissociation constant of 2.2 pM or less.3,4 TG
induced inhibition of the SERCA pump leads to deple-
tion of the ER Ca2+ pool and a capacitance influx of
extracellular Ca2+ resulting in a sustained elevation
(i.e., 200-400 nM) of the cytosolic Ca2+ concentration
([Ca2+]i).5 This sustained elevation of [Ca2+]i subse-
quently leads to DNA fragmentation and programmed
cell death (apoptosis) of treated cells.
TG induces apoptosis in rat (AT-3) and human (TSU-
Pr1, PC-3, DU-145) androgen-independent prostatic
cancer cell lines with LC50 values for cell death in the
10-100 nM range.5 This TG induced apoptosis does not
require the cells to be in proliferative cell cycle but can
be induced in primary human prostatic cancer cell
cultures in which about 98% of the cells are out of cycle
in G0.6 These studies have identified the SERCA pump
as a new therapeutic target for activating apoptosis of
androgen-independent prostatic cancer cells.
a
Structures of thapsigargin (TG, 1) and O-8-debutanoylthap-
sigargin (DBTG, 2).
be difficult to administer TG systemically as a thera-
peutic agent without significant host toxicity. One
approach to specifically target TG cytotoxicity to pros-
tatic cancer cells is to take advantage of the unique
secretion of prostate-specific antigen (PSA) by these
cells. PSA is a serine protease with chymotrypsin-like
substrate specificity that is enzymatically active only
in the extracellular fluid of prostatic cancer cells,
whereas it is enzymatically inactivated in blood serum.7-9
Previously a highly specific and efficient PSA substrate
with the sequence His-Ser-Ser-Lys-Leu-Gln-
(HSSKLQ) was identified.9 If TG is converted to O-8-
debutanoylthapsigargin (DBTG, 2) (Chart 1) and DBTG
is esterified in the O-8 position with an amino carboxylic
acid linker, the resulting TG analogue can be coupled
to the C-terminal glutamine (Q) of this peptide to form
a peptide bond that is hydrolyzable by enzymatically
active PSA (Figure 1). Such a TG analogue prodrug will
be cleaved only in the extracellular fluid of PSA-
TG’s ability to kill proliferatively quiescent G0 cells
by inhibiting the ubiquitous SERCA’s means that it will
* To whom correspondence should be addressed. Department of
Medicinal Chemistry, The Royal Danish School of Pharmacy,
2
Universitetsparken, DK-2100 Copenhagen, Denmark. Phone: (+45)
35 30 62 53. Fax: (+45) 35 30 60 41. E-mail: sbc@dfh.dk.
† The Royal Danish School of Pharmacy.
‡ J ohns Hopkins Oncology Center.
§ The Royal Veterinary and Agricultural University.
10.1021/jm010985a CCC: $20.00 © 2001 American Chemical Society
Published on Web 11/22/2001

Thapsigargin Analogues for Targeting Apoptosis
J ournal of Medicinal Chemistry, 2001, Vol. 44, No. 26 4697
Ch a r t 2a
F igu r e 1. Prostate-specific antigen (PSA) releases the active
TG analogue from the TG analogue prodrug by hydrolysis of
the peptide promoiety (HSSKLQ).
secreting prostatic cancer cells thus specifically target-
ing the TG analogue cytotoxicity to prostatic cancer
cells.
In a previous study,10 we reported on the synthesis
and apoptotic activity of a series of O-8 substituted
aromatic amine TG analogues (3c,e,f and 4a -e) (Chart
2). Analogues 4c-e were the most potent, being 1.5, 1.7,
and 1.7 times less potent inhibitors of the rabbit skeletal
muscle SERCA pump than TG, respectively, and 9, 4,
and 8 times less cytotoxic against TSU-Pr1 human
prostatic cancer cells, respectively.
The goal of this paper was to investigate the struc-
ture-activity relationship of TG analogues, in which the
O-8 butyric acid has been substituted with an ω-amino
acid (analogues 6f-j) (Chart 2) or an R-amino acid
conjugated ω-amino acid (analogues 5 and 7k -o) (Chart
2).
a
TG analogues with amine-containing linkers esterified to the
O-8 hydroxyl of 2.
15k ,l, and NR-Boc-L-alanine was coupled to methyl ester
14j to give 15m . Deprotection of the carboxylic acid gave
16k -m , coupling to 2 gave 17k -m , and deprotection
of the amine gave 7k -m . Analogues 7n ,o were synthe-
sized as outlined in Scheme 4. NR-Boc-L-serine and NR-
Boc-L-phenylalanine were coupled to analogue 6j to give
18n ,o, and deprotection of the amine gave 7n ,o.
P h a r m a cology
SERCA containing microsomes were isolated from
rabbit skeletal muscle by differential centrifugation of
the muscle homogenate.14,15
All target compounds were evaluated for their ability
to elevate [Ca2+]i in TSU-Pr1 cells, their ability to inhibit
the rabbit skeletal muscle SERCA pump, and their
ability to kill TSU-Pr1 human prostatic cancer cells.
The SERCA activity was measured with a coupled
enzyme assay as the rate of ATP hydrolysis.16,17 The
activity at each dose (nmol/mg of SR protein) of TG
analogue was expressed as percentage of the uninhib-
ited control activity and was determined in triplicate.
Inhibition curves were corrected for Ca2+-independent
(TG insensitive) ATPase activity by subtracting the
residual activity at high inhibitor concentrations, which
typically represented 10% of the total activity. The
amount of TG analogue required to inhibit 50% of the
maximal Ca2+-dependent ATPase activity in 1 mg of SR
protein was expressed as ID50 values, and was deter-
mined by 4-parameter curve-fitting (Table 1).
Ch em istr y
DBTG (2) was prepared from TG (1) by selective
trans-esterification of the O-8-butanoyl ester in metha-
nol using triethylamine as catalyst.11,12 Analogue 5 was
synthesized as shown in Scheme 1. 4-Amino-trans-
cinnamic acid hydrochloride was hydrogenated and the
carboxyl group protected as the methyl ester to give 8.
NR-Boc-L-leucine was coupled13 to the aromatic amine
to give 9. Deprotection of the carboxylic acid gave 10,
coupling to 2 gave 11, and deprotection of the amine
gave analogue 5. Analogues 6f-j were synthesized as
outlined in Scheme 2. Coupling of the N-Boc protected
aliphatic amino acids 12f-j with 2 gave 13f-j, and
deprotection of the amino group gave 6f-j. Analogues
7k -m were synthesized as shown in Scheme 3. NR-Boc-
L-leucine was coupled to methyl esters 14f,j to give
The apoptotic activity of each TG analogue against
TSU-Pr1 human prostatic cancer cells was determined
as previously described.18 The apoptotic activity was
expressed as the concentration of analogue LC50 (µM)
capable of inducing 50% loss of clonogenic survival as
compared to untreated controls (Table 1).

4698 J ournal of Medicinal Chemistry, 2001, Vol. 44, No. 26
J akobsen et al.
Sch em e 1a
a
Reagents: (a) Pd/C, H2, 2-metoxyethanol; (b) MeOH, SOCl2; (c) NR-tert-butoxycarbonyl-L-leucine, hexachloroacetone, Ph3P, pyridine,
THF; (d) 2 M NaOH (aq), MeOH; (e) 2, DCC, DMAP, CH2Cl2; (f) TFA, CH2Cl2.
Sch em e 2a
Ta ble 1. ID50 (nmol/mg of SR protein) Values for Inhibiting
50% of Maximal Rabbit Skeletal Muscle SERCA Activity and
LC50 (µM) Values for 50% Loss of Clonogenic Survival of
Human Prostate Cancer TSU-Pr1 Cellsa
ID50
(nmol/mg
SR protein)
activity
relative
to TGb
activity
relative
to TGc
LC50
(µM)
compd
TG
5
6f
6g
6h
6i
6j
7k
7l
7m
7n
7o
13.4 ( 1.4
466 ( 22
1332 ( 83
1206 ( 57
223 ( 15
40 ( 3
1
0.03 ( 0.004
0.88 ( 0.04
>20
10.92 ( 2.28
3.85 ( 0.21
0.75 ( 0.03
1.16 ( 0.16
>20
0.03 ( 0.01
0.28 ( 0.06
0.89 ( 0.04
0.21 ( 0.02
1
29
>667
364
128
25
35
99
90
17
3.0
2.6
287
3.4
1.2
0.8
-
a
Reagents: (a) 5 M NaOH (aq), (Boc)2O, tert-BuOH; (b) 2, DCC,
DMAP, CH2Cl2; (c) TFA, CH2Cl2.
35 ( 4
39
3842 ( 315
45 ( 3
16.5 ( 1.6
10.3 ( 0.5
n.d.
>667
1.0
9.3
30
The increase in cytosolic calcium concentration ([Ca2+]i)
in TSU-Pr1 cells induced by TG analogues at effective
cytotoxic concentrations was determined as previously
described (Table 2).10,19
7.0
a
Results expressed as mean ( standard deviation of triplicate
measurements. b ID50 analogue/ID50 TG. c LC50 analogue/LC50 TG.
Resu lts a n d Discu ssion
Previous published results concerning PSA activated
doxorubicin prodrugs20 promoted us to conjugate ana-
logue 4c with L-leucine to give analogue 5. The conju-
gated analogue showed decreased SERCA inhibition and
apoptotic activity (Table 1). The decreased activity of 5
was attributed to a decreased lipophilicity due to
protonization of the R-amine at physiological pH. Previ-
ously, a positive correlation between lipophilicity and
histamine-releasing activity was found in a series of O-2
and O-8 substituted TG analogues.21
The previously prepared aromatic analogues 3c,e,f
were 328, 81, and 4.4 times less potent SERCA inhibi-
tors than TG, respectively, and >800, 533, and 103
times less cytotoxic, respectively.10 Apparently, the
potency also in this case increases with increasing
lipophilicity. A similar structure-activity relationship
was found in the series of aliphatic analogues 6f-j
(Table 1).
Conjugation of 6f with L-leucine to give 7k decreased
the SERCA inhibitory activity, and the apoptotic activity
was still poor (Table 1). In contrast, conjugation of 6j
with L-leucine to give 7l only marginally changed the
SERCA inhibition, and surprisingly increased the apo-
ptotic activity affording an analogue equipotent with
TG. Replacement of L-leucine in 7l with L-alanine and
L-phenylalanine (7m ,o, respectively) only to a limited
extent influenced the activity, whereas introduction of
the more hydrophilic L-serine (7n ) afforded a less
apoptotic analogue.
Sch em e 3a
a
Reagents: (a) MeOH, SOCl2; (b) NR-tert-butoxycarbonyl-L-leucine or NR-tert-butoxycarbonyl-L-alanine, DIPEA, DCC, CH2Cl2; (c) 2 M
NaOH (aq), MeOH; (d) 2, DCC, DMAP, CH2Cl2; (e) TFA, CH2Cl2.

Thapsigargin Analogues for Targeting Apoptosis
J ournal of Medicinal Chemistry, 2001, Vol. 44, No. 26 4699
Sch em e 4a
R, â, and γ. Signals originating from the TG nucleus of all the
O-8 acylated compounds were in the following range: 1H NMR
(CDCl3) δ 0.86-0.87 (t, J ) 7-8 Hz, 3H, octanoyl H-8), 1.26-
1.28 (m, 8H, octanoyl H-4 to H-7), 1.37-1.44 (br s, 3H, H-14),
1.39-1.50 (s, 3H, H-13), 1.58-1.60 (m, 2H, octanoyl H-3),
1.83-1.87 (br s, 3H, H-15), 1.88-1.90 (s, 3H, acetyl H-2), 1.90-
1.92 (m, 3H, angeloyl C-2 CH3), 1.99-2.00 (d, J ) 7-8 Hz,
3H, angeloyl H-4), 2.28-2.30 (m, 2H, octanoyl H-2), 2.28-2.42
(dd, J ) 3-4 and 13-15 Hz, 1H, H-9′), 2.89-3.03 (dd, J )
3-4 and 13-15 Hz, 1H, H-9), 4.18-4.33 (br s, 1H, H-1), 5.46-
5.51 (m, 1H, H-2), 5.61-5.71 (m, 3H, H-3, H-6 and H-8), 6.10-
6.12 (q, J ) 7-8 Hz, 1H, angeloyl H-3). Some times H-8
appeared at a slightly lower ppm value than H-3 and H-6 and
some times H-9′ was overlapped by signals from the acyl
residues. 13C NMR (CDCl3) (labeled assignments are inter-
changeable) δ 12.8-13.4 (C-15), 14.1-14.4 (octanoyl C-8),
15.7-16.1 (C-13), 15.8-16.3 (angeloyl C-4), 19.6-20.8 (an-
geloyl C-2 CH3), 22.2-22.8 (acetyl C-2), 22.5-22.8 (C-14),
23.6-24.4 (octanoyl C-7), 24.3-25.2 (octanoyl C-3), 29.0-29.5
(octanoyl C-4 to C-6), 34.6-36.1 (octanoyl C-2), 38.3-40.7 (C-
9), 57.5-59.4 (C-1), 66.2-66.5 (C-8)a, 77.0-77.8 (C-6)a, 77.9-
78.8 (C-2)a, 78.5-78.9 (C-7)b, 78.6-79.0 (C-11)b, 84.3-85.7 (C-
3)a, 84.7-85.7 (C-10), 127.7-127.9 (angeloyl C-2), 130.1-131.2
(C-4)c, 138.8-139.2 (angeloyl C-3), 141.1-142.1 (C-5)c, 165.1-
167.4 (CdO, angeloyl), 170.9-171.8 (CdO, acetyl), 172.9-
173.1 (CdO, octanoyl), 174.5-177.6 (CdO, C-12). The identity
of target compounds was confirmed with NMR and HRMS.
The target compounds were pure according to TLC, and their
NMR spectra showed no foreign signals other than minor
solvent residuals.
a
Reagents: (a) NR-tert-butoxycarbonyl-L-serine or NR-tert-bu-
toxycarbonyl-L-phenylalanine, DCC, HOBT, DMF; (b) TFA, CH2Cl2.
Ta ble 2. Increase in Cytosolic Ca2+ Concentration ([Ca2+]i) in
TSU-Pr1 Cells at Effective Cytotoxic Concentrationsa
1000 nM [Ca2+
(nM)
]
i
100 nM [Ca2+
(nM)
]
i
compd
5
451 ( 54
284 ( 51
209 ( 6
217 ( 20
420 ( 3
369 ( 2
84 ( 7
414 ( 44
410 ( 45
348 ( 20
446 ( 33
91 ( 16
NEb
6f
6g
6h
6i
6j
7k
7l
7m
7n
7o
NEb
NEb
242 ( 7
235 ( 12
40 ( 3
173 ( 28
338 ( 10
93 ( 10
406 ( 15
8-O-Debu ta n oylth a p siga r gin (2). Triethylamine (2.5 mL)
was added to a solution of 1 (0.80 mmol) in dry MeOH (50
mL) at room temperature. After 6 h at room temperature, the
mixture was concentrated in vacuo. The residue was concen-
trated two times from toluene (50 mL) in vacuo to give 2 (yield
100%) as a white amorphous solid: 1H NMR (CDCl3) δ 0.87
(t, J ) 7.0 Hz, 3H, octanoyl H-8), 1.28 (br s, 8H, octanoyl H-4
to H-7), 1.44 (s, 3H, H-14), 1.49 (s, 3H, H-13), 1.60 (m, 2H,
octanoyl H-3), 1.84 (s, 3H, H-15), 1.90 (m, 6H, acetyl H-2 and
angeloyl C-2 CH3), 1.99 (d, J ) 7.0 Hz, 3H, angeloyl H-4), 2.29
(m, 2H, octanoyl H-2), 2.47 (dd, J ) 3.3 and 14.1 Hz, 1H, H-9′),
2.84 (d, J ) 14.1 Hz, 1H, H-9), 3.57 (br s, 1H, H-8), 4.36 (s,
1H, H-1), 5.45 (m, 1H, H-2), 5.69 (s, 1H, H-3), 5.80 (s, 1H, H-6),
6.12 (q, J ) 7.2 Hz, 1H, angeloyl H-3).
3-(4-Am in op h en yl)p r op ion ic Acid Meth yl Ester (8).
Triethylamine (10.0 mmol) was added dropwise to a suspen-
sion of 4-amino-trans-cinnamic acid hydrochloride (10.0 mmol)
in 2-methoxyethanol (12 mL) at room temperature. The
suspension was filtered, and Pd-C 10% (120 mg) was added
to the filtrate. The mixture was hydrogenated (4 atm) for 3 h
at room temperature. The mixture was filtered through a
column of Celite, and the filtrate was concentrated in vacuo
to give 3-(4-aminophenyl)propionic acid (1.7 g) as a yellowish
crystalline solid. This compound was esterified without further
purification. Thionyl chloride (2.5 mL) was added dropwise to
dry MeOH (10 mL) at -10 °C, and the solution was left for 10
min. 3-(4-Aminophenyl)propionic acid (1.7 g) was added to the
solution, and the mixture was left overnight at room temper-
ature. The solvent was removed in vacuo, and the residue was
dissolved in EtOAc (200 mL). The solution was washed with
5% NaHCO3 (200 mL), 10% NaCl (100 mL), and water (100
mL). The organic phase was dried (MgSO4), filtered, and
concentrated in vacuo to give 8 (yield 80%) as a crystalline
yellow solid: 1H NMR (CDCl3) δ 2.55 (t, J ) 7.0 Hz, 2H, H-2),
2.85 (t, J ) 7.0 Hz, 2H, H-3), 3.65 (s, 3H, OCH3), 6.60 (d, J )
9.0 Hz, 2H, Ar H-3 and H-5), 6.95 (d, J ) 9.0 Hz, 2H, Ar H-2
and H-6).
a
[Ca2+]i was monitored for 20 min. Baseline [Ca2+]i was 35 (
4 nM. Results are expressed as the mean ( standard error of
triplicate measurements. NE ) no significant elevation of [Ca2+
]
i
b
above baseline level.
The ability of the analogues to elevate [Ca2+]i in TSU-
Pr1 cells followed the relative SERCA inhibitory and
apoptotic activities (Table 2).
In conclusion, a series of TG analogues containing an
R-amino acid applicable for conjugation to PSA-specific
peptides has been prepared. In general, the potency
follows the relative lipophilicity of the analogues. The
high activity of 7l makes this analogue especially
interesting as a drug moiety in a prodrug of TG.
Exp er im en ta l Section
Ch em istr y. TG (1) was isolated from the seeds of Thapsia
garganica L. as previously described.1 Reagents and precursors
were supplied by Aldrich and were used without further
purification. Thin-layer chromatography was done using pre-
coated aluminum sheets with Silica gel 60 F254 or RP-18 F254
(Merck). Compounds were visualized by inspection under UV
(λ ) 254 nm) or after spraying with naphthoresorcinol solution
(0.2% w/v in ethanol diluted 1:1 with 2 M H2SO4) or ninhydrin
solution (0.05% w/v in ethanol) followed by heating. Normal
phase column chromatography (NPCC) was done with Silica
gel 60, 40-63 µm (Merck) using mixtures of EtOAc-toluene-
AcOH, 20:10:0.3 (A), heptane-acetone-AcOH, 7:3:0.1 (B), or
toluene-acetone, 19:1 (C), and 1:1 (D). Reverse phase column
chromatography (RPCC) was done with LiChroprep RP-18,
40-63 µm (Merck) using mixtures of MeOH-water, 4:1 (E),
5:1 (F), 6:1 (G), 7:1 (H), and 9:1 (I) or MeOH-water-AcOH,
9:1:0.1 (J ). Melting points were measured with capillary tubes
in an oil bath and were corrected. 1H NMR (300 MHz) and
13C NMR (75 MHz) spectra were recorded on a GEMINI 2000
BB, 300 MHz spectrometer. Chemical shift values (δ) are
expressed in ppm relative to tetramethylsilane as internal
standard. The following abbreviations are used for multiplicity
of NMR signals: br ) broad, s ) singlet, d ) doublet, t )
triplet, q ) quartet, dd ) double doublet, m ) multiplet. NMR
signals corresponding to exchangeable protons are omitted.
Signals from R-amino acids are assigned with Greek letters
3-(4-[NR-ter t-Bu toxycar bon yl-L-leu cin oylam in o]ph en yl)-
p r op a n oic Acid Meth yl Ester (9). A solution of triphenyl-
phosphine (1.00 mmol) in dry THF (1.0 mL) was under argon
dropwise added to a solution of NR-tert-butoxycarbonyl-L-
leucine (1.00 mmol) and hexachloroacetone (0.50 mmol) in dry
THF (2.0 mL) at -78 °C, and the solution was left for 30 min.
A solution of compound 8 (1.00 mmol) in dry THF (1.0 mL)

4700 J ournal of Medicinal Chemistry, 2001, Vol. 44, No. 26
J akobsen et al.
and a solution of pyridine (6.00 mmol) in dry THF (6.0 mL)
was added to the reaction mixture at -78 °C, and the mixture
was left at room temperature for 1 h and filtered. The filtrate
was concentrated in vacuo, and the residue was dissolved in
CH2Cl2 (100 mL). The solution was washed twice with 1 M
HCl (50 mL), twice with 5% NaHCO3 (50 mL), and twice with
10% NaCl (50 mL) and concentrated in vacuo. Purification of
the residue by NPCC (eluent A) afforded 9 (50%) as a yellowish
crystalline solid: 1H NMR (CDCl3) δ 0.95 (m, 6H, Leu CH3
and CH′3), 1.43 (s, 9H, Boc CH3), 1.62 (m, 1H, γ-H), 1.72 (m,
2H, â-H), 2.58 (t, J ) 7.8 Hz, 2H, H-2), 2.88 (t, J ) 7.8 Hz,
2H, H-3), 3.66 (s, 3H, OCH3), 4.35 (m, 1H, R-H), 7.07 (d, J )
8.1 Hz, 2H, Ar H-2 and H-2′), 7.41 (d, J ) 8.1 Hz, 2H, Ar H-3
and H-3′); 13C NMR (CDCl3) δ 22.3 (Leu CH3), 23.6 (Leu CH′3),
25.2 (γ-C), 28.5 (Boc CH3), 31.0 (C-3), 36.5 (C-2), 41.7 (â-C),
52.1 (OCH3), 54.5 (R-C), 81.2 (Boc tert-C), 120.3 (Ar C-3 and
C-5), 129.0 (Ar C-2 and C-6), 132.7 (Ar C-1), 136.7 (Ar C-4),
156.4 (CdO, carbamate), 171.2 (CdO, C-1), 173.4 (CdO,
amide).
C-6), 136.3 (Ar C-4), 172.0 (CdO, C-1), 176.5 (CdO, amide);
HRMS (FAB+) m/z 841.4551 ([M+H]+, C45H65N2O13 requires
841.4487).
6-ter t-Bu t oxyca r bon yla m in oh exa n oic Acid (12f). A
solution of sodium hydroxide (1.47 mmol) in water (0.3 mL)
was added to a solution of 6-aminohexanoic acid (1.50 mmol)
in tert-butyl alcohol (3.0 mL), and the solution was left for 10
min at room temperature. Di-tert-butyl-dicarbonate (1.65
mmol) dissolved in tert-butyl alcohol (2.8 mL) was added to
the solution, and the mixture was left overnight at room
temperature. The mixture was concentrated in vacuo, and the
residue was suspended in water (2.4 mL). The suspension was
cooled on ice and acidified (pH 2) with 2 M H2SO4. The aqueous
suspension was quickly extracted three times with EtOAc (3.6
mL), and the combined organic phases were washed three
times with water (2.4 mL), dried (MgSO4), filtered, and
concentrated in vacuo. Purification of the residue by NPCC
(eluent B) afforded 12f (yield 61%) as white crystals: mp 38-
1
39 °C; H NMR (CDCl3) δ 1.30 (m, 2H, H-4), 1.40 (s, 9H, Boc
CH3), 1.50 (m, 2H, H-3 or H-5), 1.57 (m, 2H, H-3 or H-5), 2.28
(t, J ) 7.4 Hz, 2H, H-2), 3.01 (m, 2H, H-6).
3-(4-[NR-ter t-Bu toxycar bon yl-L-leu cin oylam in o]ph en yl)-
p r op a n oic Acid (10). 2 M NaOH (10 mL) was added at room
temperature to a solution of 9 (4.89 mmol) in MeOH (65 mL),
and the mixture was left for 10 min at room temperature. The
MeOH was removed in vacuo, and the aqueous residue was
acidified to pH 2 with 2 M H2SO4. The aqueous solution was
extracted twice with EtOAc (60 mL), and the combined organic
phases were washed with 10% w/v NaCl (60 mL) and water
(60 mL). The organic phase was dried (MgSO4), filtered, and
concentrated in vacuo to give 10 (99%) as a yellowish crystal-
Compounds 12g-j were prepared as described for 12f using
7-aminoheptanoic acid, 8-aminooctanoic acid, 11-aminoun-
decanoic acid, and 12-aminododecanoic acid, respectively, as
starting materials.
7-ter t-Bu t oxyca r b on yla m in oh ep t a n oic Acid (12g).
NPCC (eluent A) afforded 12g (yield 82%) as white crystals:
1
mp 55-56 °C; H NMR (CDCl3) δ 1.35 (m, 4H, H-4 and H-5),
1.45 (s, 9H, Boc CH3), 1.48 (m, 2H, H-3 or H-6), 1.64 (m, 2H,
H-3 or H-6), 2.35 (t, J ) 7.5 Hz, 2H, H-2), 3.10 (m, 2H, H-7).
8-ter t-Bu toxycar bon ylam in ooctan oic Acid (12h ). NPCC
(eluent A) afforded 12h (yield 79%) as white crystals: mp 56-
1
line solid: mp 64.5-66.5 °C; H NMR (CDCl3) δ 0.93 (d, J )
6.3 Hz, 3H, Leu CH3), 0.96 (d, J ) 6.3 Hz, 3H, Leu CH′3), 1.38
(s, 9H, Boc CH3), 1.66 (m, 1H, γ-H), 1.74 (m, 2H, â-H), 2.63 (t,
J ) 7.2 Hz, 2H, H-2), 2.89 (t, J ) 7.2 Hz, 2H, H-3), 4.48 (m,
1H, R-H), 7.05 (d, J ) 8.0 Hz, 2H, Ar H-2 and H-6), 7.43 (d, J
) 8.0 Hz, 2H, Ar H-3 and H-5); 13C NMR (CDCl3) δ 21.9 (Leu
CH3), 23.0 (Leu CH′3), 24.8 (γ-C), 28.4 (Boc CH3), 30.2 (C-3),
35.8 (C-2), 41.4 (â-C), 54.0 (R-C), 80.5 (Boc tert-C), 120.4 (Ar
C-3 and C-5), 128.9 (Ar C-2 and C-6), 136.4 (Ar C-1), 156.9
(CdO, carbamate), 176.5 (CdO, amide), 178.2 (CdO, acid); MS
(FAB+) m/z 379 ([M+H]+, 33%), 323 (100%).
1
57 °C; H NMR (CDCl3) δ 1.30 (m, 6H, H-4 to H-6), 1.45 (s,
9H, Boc CH3), 1.46 (m, 2H, H-3 or H-7), 1.63 (m, 2H, H-3 or
H-7), 2.34 (t, J ) 7.4 Hz, 2H, H-2), 3.10 (m, 2H, H-8).
11-ter t-Bu toxyca r bon yla m in ou n d eca n oic Acid (12i).
NPCC (eluent A) afforded 12i (yield 54%) as white crystals:
mp 67-68 °C; 1H NMR (CDCl3) δ 1.28 (br s, 12H, H-4 to H-9),
1.45 (br s, 9H, Boc CH3), 1.53 (m, 2H, H-3 or H-10), 1.63 (m,
2H, H-3 or H-10), 2.34 (t, J ) 7.5 Hz, 2H, H-2), 3.10 (m, 2H,
H-11).
8-O-(3-[4-{NR-ter t-Bu toxyca r bon yl-L-leu cin oyla m in o}-
p h en yl]p r op a n oyl)-8-O-d ebu ta n oylth a p siga r gin (11). To
a mixture of 10 (0.26 mmol), 2 (0.26 mmol), and DMAP (0.03
mmol) in dry CH2Cl2 (2.0 mL) was added a solution of DCC
(0.29 mmol) in dry CH2Cl2 (0.75 mL) at 0 °C. The mixture was
left for 1 h at 0 °C and then 7.5 h at room temperature. The
mixture was filtered, and the filtrate was concentrated in
vacuo. Purification of the residue by RPCC (eluent F) yielded
11 (50%) as a white amorphous solid: 3-(4-[NR-tert-butoxy-
carbonyl-L-leucinoylamino]phenyl)propanoyl 1H NMR (CDCl3)
δ 0.93 (d, J ) 6.3 Hz, 3H, Leu CH3), 0.97 (d, J ) 6.3 Hz, 3H,
Leu CH′3) 1.40 (s, 9H, Boc CH3), 1.62 (m, 1H, γ-H), 1.74 (m,
2H, â-H), 2.56 (t, J ) 7.0 Hz, 2H, H-2), 2.83 (t, J ) 7.0 Hz,
2H, H-3), 4.34 (m, 1H, R-H), 7.00 (d, J ) 8.0 Hz, 2H, Ar H-2
and H-2′), 7.35 (d, J ) 8.0 Hz, 2H, Ar H-3 and H-3′); 13C NMR
(CDCl3) δ 23.0 (Leu CH′3 and CH3), 24.8 (γ-C), 28.4 (Boc CH3),
29.8 (C-3), 36.2 (C-2), 40.9 (â-C), 53.9 (R-C), 80.6 (Boc tert-C),
120.5 (Ar C-3 and C-5), 128.7 (Ar C-2 and C-6), 136.3 (Ar C-4),
156.7 (CdO, carbamate), 172.0 (CdO, C-1), 176.5 (CdO,
amide); HRMS (FAB+) m/z 963.4902 ([M+Na]+, C50H73N2O15Na
requires 963.4830).
12-ter t-Bu toxyca r bon yla m in od od eca n oic Acid (12j).
NPCC (eluent B) afforded 12j (yield 33%) as white crystals:
mp 83.5-84.5 °C; H NMR (CDCl3) δ 1.27 (br s, 14H, H-4 to
H-10), 1.45 (br s, 9H, Boc CH3), 1.50 (m, 2H, H-3 or H-11),
1.63 (m, 2H, H-3 or H-11), 2.35 (t, J ) 7.4 Hz, 2H, H-2), 3.10
(m, 2H, H-12).
1
8-O-(6-ter t-Bu t oxyca r b on yla m in oh exa n oyl)-8-O-d e-
bu ta n oylth a p siga r gin (13f). Compound 2 (0.18 mmol), 12f
(0.20 mmol), and DMAP (0.20 mmol) was dissolved in dry CH2-
Cl2 (1.5 mL) at room temperature. After cooling on ice, a
solution of DCC (0.20 mmol) in dry CH2Cl2 (0.5 mL) was added.
The mixture was kept on ice for 1 h and then left for 5 h at
room temperature. The mixture was filtered, and the filtrate
was concentrated in vacuo. Purification of the residue by RPCC
(eluent E) afforded 13f (yield 54%) as a white amorphous
1
solid: 6-tert-Butoxycarbonylaminohexanoyl H NMR (CDCl3)
δ 1.28 (m, 2H, H-4), 1.43 (s, 9H, Boc CH3), 1.61 (m, 4H, H-3
and H-5), 2.32 (m, 2H, H-2), 3.09 (m, 2H, H-6); 13C NMR
(CDCl3) δ 25.2 (C-3), 25.8 (C-4), 28.7 (Boc CH3), 29.5 (C-5),
34.8 (C-2), 40.7 (C-6), 80.1 (Boc tert-C), 172.7 (CdO, C-1);
HRMS (FAB+) m/z 816.4139 ([M+Na]+, C41H63NO14Na re-
quires 816.4146).
8-O-(3-[4-{L-Leu cin oyla m in o}p h en yl]p r op a n oyl)-8-O-
d ebu ta n oylth a p siga r gin (5). TFA (1.00 mL) was added to
a solution of 11 (0.12 mmol) in dry CH2Cl2 (2.0 mL) at room
temperature. The mixture was stirred for 1 h at room tem-
perature and concentrated in vacuo to give 5 (yield 100%) as
a yellowish amorphous solid: 3-(4-[L-leucinoylamino]phenyl)-
propanoyl 1H NMR (CDCl3) δ 0.86 (m, 6H, Leu CH3 and CH′3),
1.59 (m, 1H, γ-H), 1.74 (m, 2H, â-H), 2.58 (br s, 2H, H-2), 2.83
(br s, 2H, H-3), 4.21 (m, 1H, R-H), 7.02 (br s, 2H, Ar H-2 and
H-6), 7.22 (br s, 2H, Ar H-3 and H-5); 13C NMR (CDCl3) δ 23.0
(Leu CH′3 and CH3), 24.8 (γ-C), 29.8 (C-3), 36.2 (C-2), 40.9 (â-
C), 53.9 (R-C), 120.5 (Ar C-3 and C-5), 128.7 (Ar C-2 and
Compounds 13g-j were prepared as described for 13f, using
compounds 12g-j, respectively, as starting materials.
8-O-(7-ter t-Bu t oxyca r b on yla m in oh ep t a n oyl)-8-O-d e-
bu ta n oylth a p siga r gin (13g). RPCC (eluent F) afforded 13g
(yield 41%) as a white amorphous solid: 7-tert-Butoxycarbonyl-
1
aminoheptanoyl H NMR (CDCl3) δ 1.28 (m, 4H, H-4 to H-5),
1.44 (s, 9H, Boc CH3), 1.60 (m, 4H, H-3 and H-6), 2.29 (m, 2H,
H-2), 3.07 (m, 2H, H-7); 13C NMR (CDCl3) δ 25.0 (C-3), 28.6
(Boc CH3), 29.0 (C-4 and C-5), 32.0 (C-6), 34.4 (C-2), 40.0 (C-
7), 80.0 (Boc tert-C), 172.9 (CdO, C-1); HRMS (FAB+) m/z
830.4419 ([M+Na]+, C42H65NO14Na requires 830.4303).

Thapsigargin Analogues for Targeting Apoptosis
J ournal of Medicinal Chemistry, 2001, Vol. 44, No. 26 4701
8-O-(8-t er t -Bu t oxyca r b on yla m in ooct a n oyl)-8-O-d e -
bu ta n oylth a p siga r gin (13h ). RPCC (eluent F) afforded 13h
(yield 52%) as a white amorphous solid: 8-tert-Butoxycarbonyl-
aminooctanoyl 1H NMR (CDCl3) δ 1.30 (m, 6H, H-4 to H-6),
1.43 (s, 9H, Boc CH3), 1.60 (m, 4H, H-3 and H-7), 2.29 (m, 2H,
H-2), 3.07 (m, 2H, H-8); 13C NMR (CDCl3) δ 24.9 (C-3), 28.4
(Boc CH3), 28.9 (C-4 to C-6), 31.6 (C-7), 34.3 (C-2), 40.3 (C-8),
79.8 (Boc tert-C), 172.9 (CdO, C-1); HRMS (FAB+) m/z
844.4340 ([M+Na]+, C43H67NO14Na requires 844.4459).
8-O-(11-ter t-Bu t oxyca r b on yla m in ou n d eca n oyl)-8-O-
d ebu ta n oylth a p siga r gin (13i). RPCC (eluent F) afforded 13i
(yield 64%) as a white amorphous solid: 11-tert-Butoxycar-
the methyl ester hydrochloride. Filtration afforded 14f (yield
84%) as white crystals: mp 118-122 °C; 1H NMR (CD3OD) δ
1.44 (m, 2H, H-4), 1.69 (m, 4H, H-3, and H-5), 2.37 (t, J ) 7.5
Hz, 2H, H-2), 2.93 (t, J ) 7.5 Hz, 2H, H-6), 3.66 (s, 3H, OCH3);
13C NMR (CD3OD) δ 25.4 (C-3), 26.9 (C-4), 28.3 (C-2), 34.5 (C-
5), 40.7 (C-6), 52.2 (OCH3), 175.9 (CdO, C-1).
12-Am in od od eca n oic Acid Meth yl Ester Hyd r och lo-
r id e (14j). Thionyl chloride (4.0 mL) was slowly added to dry
MeOH (75 mL) at -10 °C. After 10 min at -10 °C, to the
solution was added 12-aminododecanoic acid (13.93 mmol), and
the mixture was left overnight at room temperature. The
solution was concentrated in vacuo, and the residue was
dissolved in MeOH (50 mL). To the solution was added Et2O
(80 mL) to precipitate the methyl ester hydrochloride. Filtra-
tion afforded 14j (yield 93%) as white crystals: mp 160-161
1
bonylaminoundecanoyl H NMR (CDCl3) δ 1.27 (m, 12H, H-4
to H-9), 1.44 (s, 9H, Boc CH3), 1.60 (m, 4H, H-3 and H-10),
2.29 (m, 2H, H-2), 3.08 (m, 2H, H-11); 13C NMR (CDCl3) δ 25.0
(C-3), 28.6 (Boc CH3), 29.1 (C-4 to C-8), 31.8 (C-10), 34.8 (C-
2), 172.8 (CdO, C-1); HRMS (FAB+) m/z 886.5028 ([M+Na]+,
1
°C; H NMR (CD3OD) δ 1.34 (m, 14H, H-4 to H-10), 1.62 (m,
4H, H-3 and H-11), 2.31 (t, J ) 7.5 Hz, 2H, H-2), 2.91 (t, J )
7.5 Hz, 2H, H-12), 3.65 (s, 3H, OCH3); 13C NMR (CD3OD) δ
26.1 (C-3), 27.5 (C-10), 28.6, 30.2, 30.3, 30.4, 30.5, 30.6, 30.6
(C-2 and C-4 to C-9), 34.9 (C-11), 40.9 (C-12), 52.1 (OCH3),
176.3 (CdO, C-1).
C
46H73NO14Na requires 886.4929).
8-O-(12-ter t-Bu t oxyca r b on yla m in od od eca n oyl)-8-O-
d ebu ta n oylth a p siga r gin (13j). RPCC (eluent I) afforded 13j
(yield 77%) as a white amorphous solid: 12-tert-Butoxycar-
1
bonylaminododecanoyl H NMR (CDCl3) δ 1.26 (m, 14H, H-4
6-(N R-t er t -Bu t oxyca r b on yl-L-le u cin oyla m in o)h e xa -
n oic Acid Meth yl Ester (15k ). NR-tert-Butoxycarbonyl-L-
leucine (5.50 mmol), 14f (5.50 mmol), and DIPEA (5.50 mmol)
was dissolved in dry CH2Cl2 (16.5 mL) at room temperature.
To the mixture cooled on ice was added a solution of DCC (6.00
mmol) in dry CH2Cl2 (6.0 mL). After 3 h at room temperature,
the mixture was filtered, and the filtrate was concentrated in
vacuo. Purification of the residue by NPCC (eluent C) afforded
15k (yield 50%) as a yellowish oil: 1H NMR (CDCl3) δ 0.93 (d,
J ) 6.5 Hz, 3H, Leu CH3), 0.94 (d, J ) 6.5 Hz, 3H, Leu CH′3),
1.34 (m, 2H, H-4), 1.44 (s, 9H, Boc CH3), 1.51 (m, 2H, H-5),
1.64 (m, 5H, H-3, â-H and γ-H), 2.31 (t, J ) 7.5 Hz, 2H, H-2),
3.24 (m, 2H, H-6), 3.67 (s, 3H, OCH3), 4.09 (m, 1H, R-H); 13C
NMR (CDCl3) δ 22.1 (Leu CH3), 23.0 (Leu CH′3), 24.5 (γ-C),
24.8 (C-3), 26.3 (C-4), 28.4 (Boc CH3), 29.2 (C-5), 33.9 (C-2),
39.2 (â-C), 41.4 (C-6), 51.6 (OCH3), 53.2 (R-C), 80.0 (Boc tert-
C), 156.1 (CdO, carbamate), 173.0 (CdO, C-1), 174.3 (CdO,
amide); HRMS (FAB+) m/z 359.2522 ([M+H]+, C18H35N2O5
requires 359.2546).
to H-10), 1.44 (s, 9H, Boc CH3), 1.60 (m, 4H, H-3 and H-11),
2.28 (m, 2H, H-2), 3.09 (m, 2H, H-12); 13C NMR (CDCl3) δ 24.9
(C-3), 26.7 (C-10), 28.5 (Boc CH3), 29.1 (C-4 to C-9), 31.7 (C-
11), 34.6 (C-2), 41.0 (C-12), 172.8 (CdO, C-1); HRMS (FAB+)
m/z 900.5084 ([M+Na]+, C47H75NO14Na requires 900.5085).
8-O-(6-Am in oh exa n oyl)-8-O-d eb u t a n oylt h a p siga r gin
(6f). TFA (0.5 mL) was added to a solution of 13f (0.05 mmol)
in dry CH2Cl2 (3.0 mL) at room temperature. The mixture was
left for 45 min at room temperature. Evaporation in vacuo
afforded 6f (yield 100%) as an amorphous yellowish solid:
1
6-Aminohexanoyl H NMR (CDCl3) δ 1.27 (m, 2H, H-4), 1.60
(m, 4H, H-3 and H-5), 2.31 (m, 2H, H-2), 2.97 (m, 2H, H-6);
13C NMR (CDCl3) δ 24.9 (C-3), 31.8 (C-5), 34.3 (C-2), 173.0
(CdO, C-1); HRMS (FAB+) m/z 694.3809 ([M+H]+, C36H56NO12
requires 694.3802).
Compounds 6g-j were prepared as described for 6f, using
compounds 13g-j, respectively, as starting materials.
8-O-(7-Am in oh e p t a n oyl)-8-O-d e b u t a n oylt h a p siga r -
gin (6g). Amorphous yelowish solid (yield 100%): 7-Amino-
heptanoyl 1H NMR (CDCl3) δ 1.27 (m, 4H, H-4 to H-5), 1.60
(m, 4H, H-3 and H-6), 2.30 (m, 2H, H-2), 2.99 (m, 2H, H-7);
13C NMR (CDCl3) δ 25.0 (C-3), 29.0 (C-4 to C-5), 31.9 (C-6),
34.4 (C-2), 40.3 (C-7), 172.9 (CdO, C-1); HRMS (FAB+) m/z
708.3965 ([M+H]+, C37H58NO12 requires 708.3959).
8-O-(8-Am in ooct a n oyl)-8-O-d eb u t a n oylt h a p siga r gin
(6h ). Amorphous yellowish solid (yield 100%): 8-Aminooctanoyl
1H NMR (CDCl3) δ 1.28 (m, 6H, H-4 to H-6), 1.60 (m, 4H, H-3
and H-7), 2.28 (m, 2H, H-2), 3.00 (m, 2H, H-8); 13C NMR
(CDCl3) δ 24.9 (C-3), 29.0 (C-4 to C-6), 31.6 (C-7), 34.8 (C-2),
40.3 (C-8), 173.1 (CdO, C-1); HRMS (FAB+) m/z 722.4113
([M+H]+, C38H60NO12 requires 722.4116).
8-O-(11-Am in ou n d eca n oyl)-8-O-d ebu ta n oylth a p siga r -
gin (6i). Amorphous yellowish solid (yield 100%): 11-Amino-
undecanoyl 1H NMR (CDCl3) δ 1.26 (m, 12H, H-4 to H-9), 1.59
(m, 4H, H-3 and H-10), 2.29 (m, 2H, H-2), 2.97 (m, 2H, H-11);
13C NMR (CDCl3) δ 24.9 (C-3), 28.8 (C-4 to C-9), 31.6 (C-10),
34.4 (C-2), 172.9 (CdO, C-1); HRMS (FAB+) m/z 764.4655
([M+H]+, C41H66NO12 requires 764.4585).
8-O-(12-Am in od od eca n oyl)-8-O-d ebu ta n oylth a p siga r -
gin (6j). Amorphous yellowish solid (yield 100%): 12-Amino-
dodecanoyl 1H NMR (CDCl3) δ 1.27 (m, 14H, H-4 to H-10), 1.60
(m, 4H, H-3 and H-11), 2.30 (m, 2H, H-2), 3.00 (m, 2H, H-12);
13C NMR (CDCl3) δ 24.9 (C-3), 29.1 (C-4 to C-10), 31.7 (C-11),
34.3 (C-2), 172.9 (CdO, C-1); HRMS (FAB+) m/z 778.4700
([M+H]+, C42H68NO12 requires 778.4742).
Compounds 15l,m were prepared as described for 15k , using
NR-tert-butoxycarbonyl-L-leucine and NR-tert-butoxycarbonyl-
L-alanine, respectively, together with compound 14j as starting
materials.
12-(N R-t er t -B u t o x y c a r b o n y l-L-le u c in o y la m in o )d o -
d eca n oic Acid Meth yl Ester (15l). NPCC (eluent C) afforded
15l (yield 53%) as white crystals: mp 63-64 °C; 1H NMR
(CDCl3) δ 0.93 (m, 6H, Leu CH3 and CH′3), 1.26 (br s, 14H,
H-4 to H-10), 1.44 (s, 9H, Boc CH3), 1.48 (m, 3H, â-H and γ-H),
1.63 (m, 4H, H-3 and H-11), 2.31 (t, J ) 7.5 Hz, 2H, H-2), 3.23
(m, 2H, H-12), 3.67 (s, 3H, OCH3), 4.08 (m, 1H, R-H); 13C NMR
(CDCl3) δ 22.2 (Leu CH3), 22.9 (Leu CH′3), 24.8 (γ-C), 25.0 (C-
3), 26.9 (C-10), 28.4 (Boc CH3), 29.2, 29.3, 29.5 (C-4 to C-9 and
C-11), 34.2 (C-2), 39.5 (â-C), 41.4 (C-12), 51.6 (OCH3), 53.2 (R-
C), 80.1 (Boc tert-C), 156.1 (CdO, carbamate), 172.8 (CdO,
C-1), 174.7 (CdO, amide); HRMS (FAB+) m/z 443.3517
([M+H]+, C24H47N2O5 requires 443.3485).
12-(N R-t er t -B u t o x y c a r b o n y l-L-a la n in o y la m in o )d o -
d eca n oic Acid Meth yl Ester (15m ). NPCC (eluent C)
afforded 15m (yield 55%) as white crystals: 1H NMR
(CD3OD) δ 1.30 (br s, 14H, H-4 to H-10), 1.44 (s, 9H, Boc CH3),
1.49 (m, 3H, Ala CH3), 1.59 (m, 4H, H-3 and H-11), 2.31 (t, J
) 7.5 Hz, 2H, H-2), 3.17 (m, 2H, H-12), 3.65 (s, 3H, OCH3),
3.99 (m, 1H, R-H); 13C NMR (CD3OD) δ 18.6 (â-C), 26.1 (C-3),
28.0 (C-10), 28.8 (Boc CH3), 30.3, 30.5, 30.7 (C-4 to C-9 and
C-11), 34.9 (C-2), 40.4 (C-12), 51.8 (OCH3), 52.1 (R-C), 80.7 (Boc
tert-C), 158.2 (CdO, carbamate), 176.3 (CdO, amide); HRMS
(FAB+) m/z 401.3036 ([M+H]+, C21H41N2O5 requires 401.3015).
6-Am in oh exa n oic Acid Met h yl E st er Hyd r och lor id e
(14f). Thionyl chloride (4.0 mL) was slowly added to dry MeOH
(30 mL) at -10 °C. After 10 min at -10 °C to the solution was
added 6-aminohexanoic acid (15.25 mmol), and the mixture
was left overnight at room temperature. The solution was
concentrated in vacuo, and the residue was dissolved in MeOH
(15 mL). To the solution was added Et2O (60 mL) to precipitate
6-(N R-t er t -Bu t oxyca r b on yl-L-le u cin oyla m in o)h e xa -
n oic Acid (16k ). 2 M NaOH (10 mL) was added to a solution
of 15k (0.5 mmol) in MeOH (20 mL), and the mixture was left
for 40 min at room temperature. The MeOH was removed in
vacuo, and the aqueous residue was cooled on ice and acidified
to pH 2 with 2 M H2SO4. The aqueous solution was extracted

4702 J ournal of Medicinal Chemistry, 2001, Vol. 44, No. 26
J akobsen et al.
three times with EtOAc (50 mL) and the combined organic
phases were washed with 10% w/v NaCl (25 mL) and water
(25 mL). The organic phase was dried (MgSO4) and filtered.
Concentration in vacuo afforded 16k (yield 90%) as white
crystals: mp 100.5-102.5 °C; 1H NMR (CDCl3) δ 0.91 (d, J )
4.5 Hz, 3H, Leu CH3), 0.93 (d, J ) 4.5 Hz, 3H, Leu CH′3), 1.37
(m, 2H, H-4), 1.43 (s, 9H, Boc CH3), 1.51 (m, 3H, â-H and γ-H),
1.65 (m, 4H, H-3 and H-5), 2.34 (t, J ) 7.5 Hz, 2H, H-2), 3.24
(m, 2H, H-6), 4.15 (m, 1H, R-H); 13C NMR (CDCl3) δ 22.1 (Leu
CH3), 22.8 (Leu CH′3), 24.4 (γ-C), 24.8 (C-3), 26.2 (C-4), 28.4
(Boc CH3), 29.0 (C-5), 33.9 (C-2), 39.3 (â-C), 41.3 (C-6), 53.2
(R-C), 80.3 (Boc tert-C), 156.4 (CdO, carbamate), 173.3 (CdO,
amide), 177.9 (CdO, C-1); HRMS (FAB+) m/z 345.2430
([M+H]+, C17H33N2O5 requires 345.2389).
Compounds 16l,m were prepared as described for 16k , using
compounds 15l,m , respectively, as starting materials.
12-(N R-t er t -B u t o x y c a r b o n y l-L-le u c in o y la m in o )d o -
d eca n oic Acid (16l). Yellowish oil (yield 95%): 1H NMR
(CDCl3) δ 0.93 (m, 6H, Leu CH3 and CH′3), 1.27 (br s, 14H,
H-4 to H-10), 1.43 (s, 9H, Boc CH3), 1.48 (m, 3H, â-H and γ-H),
1.62 (m, 4H, H-3 and H-11), 2.35 (t, J ) 7.5 Hz, 2H, H-2), 3.23
(m, 2H, H-12), 4.13 (br s, 1H, R-H); 13C NMR (CDCl3) δ 22.1
(Leu CH3), 22.8 (Leu CH′3), 24.7 (γ-C), 24.8 (C-3), 26.6 (C-10),
28.3 (Boc CH3), 28.7, 28.9, 29.1, 29.3 (C-4 to C-9 and C-11),
34.0 (C-2), 39.5 (â-C), 41.3 (C-12), 53.1 (R-C), 80.3 (Boc tert-
C), 156.4 (CdO, carbamate), 173.1 (CdO, amide), 178.3
(CdO, C-1); HRMS (FAB+) m/z 429.3356 ([M+H]+, C23H45N2O5
requires 429.3328).
(eluent F) afforded 17m (yield 78%) as a white amorphous
solid: 12-(NR-tert-Butoxycarbonyl-L-alaninoylamino)dodecanoyl
1H NMR (CDCl3) δ 1.26 (br s, 14H, H-4 to H-10), 1.43 (s, 9H,
Boc CH3), 1.47 (m, 3H, Ala CH3), 1.60 (m, 4H, H-3 and H-11),
2.28 (m, 2H, H-2), 3.22 (m, 2H, H-12), 4.12 (m, 1H, R-H); 13C
NMR (CDCl3) δ 18.2 (â-C), 24.9 (C-3), 26.6 (C-10), 28.3 (Boc
CH3), 28.8, 29.1, 29.5 (C-4 to C-9), 31.7 (C-11), 34.5 (C-2), 50.0
(R-C), 80.6 (Boc tert-C), 173.0 (CdO, C-1 and amide); HRMS
(FAB+) m/z 949.5622 ([M+H]+, C50H81N2O15 requires 949.5637).
8-O-(6-[L-Leu cin oyla m in o]h exa n oyl)-8-O-d ebu ta n oyl-
th a p siga r gin (7k ). TFA (1.2 mL) was added to a solution of
17k (0.20 mmol) in dry CH2Cl2 (3.0 mL) at room temperature.
The mixture was left for 45 min at room temperature.
Evaporation in vacuo afforded 7k (yield 100%) as an amor-
phous yellowish solid: 6-(L-Leucinoylamino)hexanoyl 1H NMR
(CDCl3) δ 0.93 (m, 6H, Leu CH3 and CH′3), 1.28 (m, 2H, H-4),
1.60 (m, 4H, H-3 and H-5), 2.29 (m, 2H, H-2), 3.20 (m, 2H,
H-6), 3.62 (m, 1H, R-H); 13C NMR (CDCl3) δ 22.6 (Leu CH3
and CH′3), 24.8 (γ-C), 24.9 (C-3), 29.0 (C-4), 31.7 (C-5), 34.3
(C-2), 38.3 (â-C), 44.3 (C-6), 53.6 (R-C), 170.8 (CdO, C-1), 172.9
(CdO, amide); HRMS (FAB+) m/z 807.4624 ([M+H]+, C42H67
N2O13 requires 807.4643).
-
Compounds 7l,m were prepared as described for 7k , using
compounds 17l,m , respectively, as starting materials.
8-O -(12-[L-L e u c i n o y la m i n o ]d o d e c a n o y l)-8-O -d e -
bu ta n oylth a p siga r gin (7l). Amorphous yellowish solid (yield
1
100%): 12-(L-Leucinoylamino)dodecanoyl H NMR (CDCl3) δ
0.95 (m, 6H, Leu CH3 and CH′3), 1.25 (br s, 14H, H-4 to H-10),
1.61 (m, 4H, H-3 and H-11), 2.33 (m, 2H, H-2), 3.25 (m, 2H,
H-12), 4.19 (m, 1H, R-H); 13C NMR (CDCl3) δ 22.6 (Leu CH3
and CH′3), 24.6 (C-3), 24.8 (γ-C), 26.5 (C-10), 28.8-29.1 (C-4
to C-9), 31.7 (C-11), 34.4 (C-2), 38.1 (â-C), 40.5 (C-12), 53.3
(R-C), 173.1 (CdO, C-1), 174.5 (CdO, amide); HRMS (FAB+)
m/z 891.5641 ([M+H]+, C48H79N2O13 requires 891.5582).
8-O -(12-[L-Ala n i n o y la m i n o ]d o d e c a n o y l)-8-O -d e -
bu ta n oylth a p siga r gin (7m ). Amorphous yellowish solid
(yield 100%): 12-(L-Alaninoylamino)dodecanoyl 1H NMR (CDCl3)
δ 1.24 (m, 14H, H-4 to H-10), 1.53 (m, 3H, Ala CH3), 1.57 (m,
4H, H-3 and H-11), 2.30 (m, 2H, H-2), 3.20 (br s, 2H, H-12),
4.22 (br s, 1H, R-H); 13C NMR (CDCl3) δ 17.4 (â-C), 24.9 (C-3),
26.8 (C-10), 28.8, 29.3, 29.6 (C-4 to C-9), 31.8 (C-11), 34.6 (C-
2), 50.4 (R-C), 174.1 (CdO, amide); HRMS (FAB+) m/z
849.5057 ([M+H]+, C45H73N2O13 requires 849.5112).
12-(N R-t er t -B u t o x y c a r b o n y l-L-a la n in o y la m in o )d o -
d eca n oic Acid (16m ). Amorphous solid (yield 93%): 1H NMR
(CD3OD) δ 1.30 (br s, 14H, H-4 to H-10), 1.44 (s, 9H, Boc CH3),
1.49 (m, 3H, Ala CH3), 1.59 (m, 4H, H-3 and H-11), 2.27 (t, J
) 7.5 Hz, 2H, H-2), 3.18 (m, 2H, H-12), 4.00 (m, 1H, R-H); 13
C
NMR (CD3OD) δ 18.8 (â-C), 26.3 (C-3), 28.2 (C-10), 28.9 (Boc
CH3), 30.7, 30.9, 31.1 (C-4 to C-9 and C-11), 35.2 (C-2), 40.6
(C-12), 52.0 (R-C), 80.8 (Boc tert-C), 175.8 (CdO, amide), 177.8
(CdO, C-1); HRMS (FAB+) m/z 387.2807 ([M+H]+, C20H39N2O5
requires 387.2859).
8-O-(6-[NR-ter t-Bu toxyca r bon yl-L-leu cin oyla m in o]h ex-
a n oyl)-8-O-d ebu ta n oylth a p siga r gin (17k ). Compound 2
(0.36 mmol), 16k (0.36 mmol), and DMAP (0.04 mmol) was
dissolved in dry CH2Cl2 (2.0 mL) at room temperature. To the
mixture cooled on ice was added a solution of DCC (0.40 mmol)
in dry CH2Cl2 (1.0 mL). The mixture was left on ice for 1 h
and then left for 3.5 h at room temperature. The mixture was
filtered, and the filtrate was concentrated in vacuo. Purifica-
tion of the residue by RPCC (eluent E) afforded 17k (yield 69%)
as a white amorphous solid: 6-(NR-tert-Butoxycarbonyl-L-
leucinoylamino)hexanoyl 1H NMR (CDCl3) δ 0.92 (m, 6H, Leu
CH3 and CH′3), 1.28 (br s, 2H, H-4), 1.42 (br s, 9H, Boc CH3),
1.61 (m, 4H, H-3 and H-5), 2.30 (m, 2H, H-2), 3.20 (m, 2H,
H-6), 4.06 (m, 1H, R-H); 13C NMR (CDCl3) δ 22.6 (Leu CH3
and CH′3), 24.8 (γ-C), 24.9 (C-3), 28.4 (Boc CH3), 29.1 (C-4),
31.7 (C-5), 34.3 (C-2), 38.4 (â-C), 41.2 (C-6), 53.1 (R-C), 80.1
(Boc tert-C) 156.3 (CdO, carbamate), 172.9 (CdO, C-1), 173.6
8-O-(12-[NR-ter t-Bu toxyca r bon yl-L-ser in oyla m in o]d o-
d eca n oyl)-8-O-d eb u t a n oylt h a p siga r gin (18n ). N-tert-
Butoxycarbonyl-L-serine (0.18 mmol), 6j (0.18 mmol), and
HOBT (0.18 mmol) were dissolved in dry DMF (2.0 mL) at
room temperature. To the mixture cooled on ice was added a
solution of DCC (0.18 mmol) in dry DMF (1.0 mL). The mixture
was left on ice for 1 h and then left for 3.5 h at room
temperature. The mixture was filtered, and the filtrate was
concentrated in vacuo. Purification of the residue by RPCC
(eluent J ) afforded 18n (yield 72%) as a white amorphous
solid: 12-(NR-tert-Butoxycarbonyl-L-serinoylamino)dodecanoyl
1H NMR (CDCl3) δ 1.27 (br s, 14H, H-4 to H-10), 1.45 (s, 9H,
Boc CH3), 1.60 (m, 4H, H-3 and H-11), 2.29 (m, 2H, H-2), 3.24
(t, J ) 6.2 Hz, 2H, H-12), 3.64 (m, 1H, â-H′), 4.05 (dd, J ) 3.0
and 11.1 Hz, 1H, â-H), 4.11 (m, 1H, R-H); 13C NMR (CDCl3)
24.9 (C-3), 26.6 (C-10), 28.3 (Boc CH3), 28.8-29.3 (C-4 to C-9),
31.7 (C-11), 34.3 (C-2), 39.6 (C-12), 62.8 (â-C), 80.8 (Boc tert-
C), 156.6 (CdO, carbamate), 171.6 (CdO, C-1), 173.2 (CdO,
amide); HRMS (FAB+) m/z 965.5593 ([M+H]+, C50H81N2O16
requires 965.5586).
(CdO, amide); HRMS (FAB+) m/z 907.5177 ([M+H]+, C47H75
N2O15 requires 907.5167).
-
Compounds 17l,m were prepared as described for 17k , using
compounds 16l,m , respectively, as starting materials.
8-O-(12-[NR-ter t-Bu toxyca r bon yl-L-leu cin oyla m in o]d o-
d eca n oyl)-8-O-d ebu ta n oylth a p siga r gin (17l). RPCC (elu-
ent D) afforded 17l (yield 94%) as a white amorphous solid:
12-(NR-tert-Butoxycarbonyl-L-leucinoylamino)dodecanoyl 1H
NMR (CDCl3) δ 0.92 (m, 6H, Leu CH3 and CH′3), 1.26 (br s,
14H, H-4 to H-10), 1.42 (br s, 9H, Boc CH3), 1.61 (m, 4H, H-3
and H-11), 2.28 (m, 3H, H-2), 3.20 (m, 2H, H-12), 4.05 (m, 1H,
R-H); 13C NMR (CDCl3) δ 22.6 (Leu CH3 and CH′3), 24.8 (γ-C),
24.9 (C-3), 26.7 (C-10), 28.4 (Boc CH3), 29.0-29.3 (C-4 to C-9),
31.7 (C-11), 34.3 (C-2), 38.4 (â-C), 41.1 (C-12), 53.1 (R-C), 80.1
(Boc tert-C), 156.2 (CdO, carbamate), 172.9 (CdO, C-1), 173.0
(CdO, amide); HRMS (FAB+) m/z 1013.5938 ([M+Na]+,
Compound 18o was prepared as described for 18n , using
NR-tert-butoxycarbonyl-L-phenylalanine as starting material.
8-O-(12-[NR-ter t-Bu t oxyca r b on yl-L-p h en yla la n in oyl-
a m in o]d od eca n oyl)-8-O-d ebu t a n oylt h a p siga r gin (18o).
RPCC (eluent J ) afforded 18o (yield 73%) as a white amor-
phous solid: 12-(NR-tert-Butoxycarbonyl-L-phenylalaninoyl-
1
amino)dodecanoyl H NMR (CDCl3) δ 1.26 (br s, 14H, H-4 to
H-10), 1.38 (br s, 9H, Boc CH3), 1.58 (m, 6H, H-3 and H-11),
2.28 (m, 3H, H-2), 3.01 (m, 2H, â-H), 3.13 (m, 2H, H-12), 4.25
(m, 1H, R-H), 7.18-7.29 (m, 5H, Ph); 13C NMR (CDCl3) δ 24.9
(C-3), 26.7 (C-10), 28.3 (Boc CH3), 29.0-29.3 (C-4 to C-9), 31.7
C
53H86N2O15Na requires 1013.5926).
8-O-(12-[NR-ter t-Bu t oxyca r b on yl-L-a la n in oyla m in o]-
d od eca n oyl)-8-O-d eb u t a n oylt h a p siga r gin (17m ). RPCC

Thapsigargin Analogues for Targeting Apoptosis
J ournal of Medicinal Chemistry, 2001, Vol. 44, No. 26 4703
(C-11), 34.3 (C-2), 38.4 (â-C), 41.1 (C-12), 62.0 (R-C), 81.4 (Boc
tert-C), 127.0 (Phe C-4), 128.8 (Phe C-2, C-2′), 129.5 (Phe C-3,
C-3′), 137.0 (Phe C-1), 158.3 (CdO, carbamate), 172.9 (CdO,
C-1); HRMS (FAB+) m/z 1025.606 ([M+H]+, C56H85N2O15
requires 1025.595).
1:100 in buffer A before making serial dilutions in buffer A.
The amount of DMSO present did not influence the measured
ATPase activity. The total ATPase activity was 7.0 µmol of
ATP (mg of SR protein)-1 min-1
.
Compounds 7n ,o were prepared as described for 7k , using
compounds 18n ,o, respectively, as starting materials.
Ack n ow led gm en t. CapCure foundation, National
Cancer Institute, Frederick, MD, and The Danish
Cancer Society are acknowledged for financial support.
8-O-(12-[L-Ser in oylam in o]dodecan oyl)-8-O-debu tan oyl-
th a p siga r gin (7n ). Amorphous yellowish solid (yield 100%):
12-(L-Serinoylamino)dodecanoyl 1H NMR (CDCl3) δ 1.26 (br
s, 14H, H-4 to H-10), 1.58 (m, 4H, H-3 and H-11), 2.29 (m,
2H, H-2), 3.17 (br s, 2H, H-12), 3.74 (dd, J ) 13.7 and 6.8 Hz,
1H, â-H′), 3.88 (br s, 1H, R-H), 4.01 (br s, 1H, â-H); 13C NMR
(CDCl3) δ 24.9 (C-3), 26.6 (C-10), 28.8-29.1 (C-4 to C-9), 31.7
(C-11), 34.3 (C-2), 55.4 (R-C), 59.6 (â-C), 173.0 (CdO, C-1),
174.4 (CdO, amide); HRMS (FAB+) m/z 865.5010 ([M+H]+,
C45H73N2O14 requires 865.5062).
Refer en ces
(1) Rasmussen, U.; Christensen, S. B.; Sandberg, F. Thapsigargin
and thapsigargicine, two new histamine liberators from Thapsia
garganica L. Acta Chem. Suec. 1978, 15, 133-140.
(2) Christensen, S. B.; Andersen, A.; Smitt, U. W. Sesquiterpenoids
from Thapsia species and medicinal chemistry of the thapsigar-
gins. Prog. Chem. Natl. Prod. 1997, 71, 129-167.
(3) Davidson, G. A.; Varhol, R. J . Kinetics of thapsigargin-Ca2+
-
ATPase (sarcoplasmic reticulum) interaction reveals a two-step
binding mechanism and picomolar inhibition. J . Biol. Chem.
1995, 270, 11731-11734.
8-O-(12-[L-P h en yla la n in oyla m in o]d od eca n oyl)-8-O-d e-
bu tan oylth apsigar gin (7o). Amorphous yellowish solid (yield
100%): 12-(L-Phenylalaninoylamino)dodecanoyl 1H NMR
(CDCl3) δ 1.27 (m, 14H, H-4 to H-10), 1.60 (m, 6H, H-3 and
H-11), 2.28 (m, 2H, H-2), 2.68 (dd, J ) 9.3 and 13.7 Hz, 1H,
â-H), 3.23 (m, 3H, â-H′ and H-12), 3.58 (dd, J ) 9.3 and 4.2
Hz, 1H, R-H), 7.20-7.34 (m, 5H, Ph); 13C NMR (CDCl3) δ 24.9
(C-3), 26.7 (C-10), 29.0-29.3 (C-4 to C-9), 31.7 (C-11), 34.3 (C-
2), 38.4 (â-C), 41.1 (C-12), 61.1 (R-C), 127.0 (Phe C-4), 128.9
(Phe C-2, C-2′), 129.5 (Phe C-3, C-3′), 138.1 (Phe C-1), 172.9
(CdO, C-1); HRMS (FAB+) m/z 925.539 ([M+H]+, C51H77N2O13
requires 925.543).
Isola tion of Sa r cop la sm ic Reticu lu m (SR). Frozen rab-
bit muscle was purchased from Pel-Freez Biologicals (Rogers,
AR) and MOPS, sucrose, EDTA, and KCl were purchased from
SIGMA. Homogenization was done with a commercial blender
(Waring, USA). Centrifugation was done with a Sorvall RC-
5B superspeed centrifuge (DuPont, USA) and a L7 ultracen-
trifuge (Beckman Coulter, USA). The temperature was kept
at 0-4 °C during the preparation. Frozen rabbit muscle (180
g) was blended 15 s every 5 min during 1 h with 510 mL of a
solution containing 10 mM MOPS, pH 7.0, 10% sucrose and
0.1 mM EDTA. The pH was kept between 6.5 and 7.0 by
adding 10% NaOH. The homogenate was centrifuged at
15000g for 20 min. The supernatant was filtered through a
path of cheesecloth, and centrifuged at 40000g for 90 min. The
pellet was suspended with a Dounce glass homogenizer in 60
mL of a solution containing 10 mM MOPS, pH 7.0, and 0.6 M
KCl. After incubating for 40 min at 4 °C, the suspension was
centrifuged at 15000g for 20 min. The 10% top of the
supernatant and the pellet were discarded. The supernatant
was collected and centrifuged at 40000g for 90 min. The pellet
was suspended with a Dounce glass homogenizer in 40 mL of
microsome storage solution containing 10 mM MOPS, pH 7.0
and 30% sucrose. The microsomes were stored at -80 °C. The
sarcoplasmic reticulum (SR) protein concentration (1.1 mg/mL)
was determined with the Micro BCA Protein Assay Reagent
kit supplied by Pierce (Rockford, IL) using bovine serum
albumin (BSA) as standard.
Mea su r em en t of ATP a se Activity. KCl, Trizma-HCl,
MgCl2, EGTA, CaCl2, â-NADH, phosphoenolpyruvate (PEP),
A23187, phosphoenolpyruvate kinase (PK), lactate dehydro-
genase (LDH), and ATP were supplied by Sigma. The ATPase
activity was measured with a Spectramax Plus384 microplate
spectrophotometer (Molecular Devices Corporation, Sunnyvale,
CA) as the rate of ATP hydrolysis essentially as previously
described.66,128 Buffer A: 0.1 M KCl, 20 mM Trizma-HCl, pH
7.5, 5 mM MgCl2, 0.5 mM EGTA, 0.7 mM CaCl2. Solution 1:
1.2 mM â-NADH, 1.5 mM PEP, 4.5 µM A23187, 22.5 U/mL
PK, 54 U/mL LDH and 30 µg/mL SR protein in buffer A.
Solution 2: Control or inhibitor dilutions in buffer A (concen-
trations corrected for a 1:3 dilution). Solution 3: 0.72 mM ATP
in buffer A. 100 µL of solution 1 was mixed with 100 µL of
solution 2 and 100 µL of solution 3 was added to start the
reaction. After 5 min of incubation, the OD340 was measured
kinetically at room temperature (n ) 3) for at least 10 min.
Typically, a 1 mM DMSO solution of inhibitor was diluted
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J M010985A