14-Aminocamptothecins: Their synthesis, preclinical activity, and potential use for cancer treatment

Article abstract of DOI:10.1021/jm101354u

14-Aminocamptothecins were synthesized in good yields by treating camptothecin (1a) and 7-ethylcamptothecin (1b) with 90% fuming nitric acid either neat or in acetic anhydride and then followed by reduction of the resulting 14-nitrocamptothecins (2). 14-Aminocamptothecin (3a) and 7-ethyl-14-aminocamptothecin (3b) demonstrated excellent cytotoxic potency against human tumor cell ines in vitro, and they are not substrates for any of the major clinically relevant efflux pumps (MDR1, MRP1, and BCRP). 3a and 3b showed similar cytotoxicity against human and mouse bone marrow progenitor cells. This is in contrast to many camptothecin analogues, which are substrates for efflux pumps and are dramatically more toxic to human marrow cells relative to murine. 3a and 3b demonstrated significant brain penetration when dosed orally in mice. 3b showed significantly better efficacy relative to topotecan when dosed orally in the three ectopic xenograft models, H460, HT29, and PC-3. On the basis of its favorable in vitro and in vivo profile, 3b warrants future development.

Full text of DOI:10.1021/jm101354u

ARTICLE
pubs.acs.org/jmc
14-Aminocamptothecins: Their Synthesis, Preclinical Activity, and
Potential Use for Cancer Treatment
Jian-Xin Duan,* Xiaohong Cai, Fanying Meng, Jessica D. Sun, Qian Liu, Donald Jung, Hailong Jiao,
Jackson Matteucci, Brian Jung, Deepthi Bhupathi, Dharmendra Ahluwalia, Heli Huang, Charles P. Hart,
and Mark Matteucci
Threshold Pharmaceuticals, 1300 Seaport Blvd, Suite 500, Redwood City, California 94063, United States
S Supporting Information
b
ABSTRACT: 14-Aminocamptothecins were synthesized in good yields
by treating camptothecin (1a) and 7-ethylcamptothecin (1b) with 90%
fuming nitric acid either neat or in acetic anhydride and then followed by
reduction of the resulting 14-nitrocamptothecins (2). 14-Aminocamp-
tothecin (3a) and 7-ethyl-14-aminocamptothecin (3b) demonstrated
excellent cytotoxic potency against human tumor cell lines in vitro, and
they are not substrates for any of the major clinically relevant efflux pumps
(MDR1, MRP1, and BCRP). 3a and 3b showed similar cytotoxicity against human and mouse bone marrow progenitor cells. This is
in contrast to many camptothecin analogues, which are substrates for efflux pumps and are dramatically more toxic to human
marrow cells relative to murine. 3a and 3b demonstrated significant brain penetration when dosed orally in mice. 3b showed
significantly better efficacy relative to topotecan when dosed orally in the three ectopic xenograft models, H460, HT29, and PC-3.
On the basis of its favorable in vitro and in vivo profile, 3b warrants future development.
’ INTRODUCTION
tetrafluoroborate in acetic anhydride, resulting in 20-O-acetyl-
14-nitrocamptothecin being obtained at a 28% yield.9 We found
that nitration of 1b went smoothly in acetic anhydride with 90% nitric
acid at 0 °C, with 2b being obtained as the only product (Scheme 1).
This is in contrast to the completely different regiochemistry reported
by Wani and Wall. The assignment of regiochemistry was based on the
1H NMR spectra of the nitrocamptothecins. The 1H NMR spectra of
1b and 2b are shown in Figure 2. The chemical shift of H-C14 of 1b
appears at 7.33 ppm as a singlet in DMSO-d6, but this peak
Camptothecin was first isolated in 1966 by Wani and Wall.1 Its
potent cytotoxicity toward tumor cells and its discovery as an inhibitor
of topoisomerase I, an essential enzyme for topological DNA mod-
ification during a number of critical cellular process,2 has stimulated
extensive analogue investigations. Two camptothecin analogues, to-
potecan (7)3 and irinotecan (8) (Figure 1),4 are currently approved
for the treatment of a variety of solid tumors, and many other
analogues are currently in clinical trials. There continue to be active
efforts to develop new camptothecin analogues that avoid the
deficiencies of the currently approved drugs, such as being substrates
for drug efflux pumps and having severe toxicity on human bone
marrow progenitor cells.5 The extensive historical SAR efforts have
largely focused on the A, B, and E rings of camptothecin. Relatively few
analogues of the D ring have been investigated; only one example at
the 14 position with biological testing has been reported.6,7 These data
showed the 14-chloro derivative7 was much less potent than the parent
camptothecin, suggesting a lack of tolerance for substitution at that
position. We developed a highly regioselective process for the synthesis
of 14-nitro and 14-aminocamptothecin and their analogues and found
that previously unknown 14-aminocamptothecins were surprisingly
potent having approximately equal potency to the unsubstituted 14
position parent camptothecins.
1
disappeared in the H NMR spectrum of the corresponding nitro
substituted product 2b. The molecular weight of the product is
consistent with 2b. On the basis of this information, the product 2b
was identified as 7-ethyl-14-nitrocamptothecin.
The different regioselectivity observed with this procedure
may be due to the fact that sulfuric acid (pKa = -3) is a very strong
acid and is able to protonate the carbonyl group in the D ring in
addition to protonation of the nitrogen in the B ring, thus
deactivating the electrophilic substitution of the D ring (Scheme 1).
Reduction of 2a and 2b with H2 in the presence of 10% Pd/C
in methanol went smoothly at room temperature, and 3a and 3b
were obtained in good yields (Scheme 2).
’ RESULTS AND DISCUSSION
Cytotoxicity Studies. The compounds were screened for
cytotoxicity using the H460 human non-small-cell lung cancer
’ CHEMISTRY
Nitration of 1a with a nitric and sulfuric acid mixture was first
reported in 1986 by Wani and Wall8 with a mixture of 9- and 12-
substituted camptothecins being formed. The 14 position has
previously been nitrated by using a large excess of nitronium
Received: October 19, 2010
Published: February 22, 2011
r
2011 American Chemical Society
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(NSCLC) cell line, 1a, 7-ethyl-10-hydroxycamptothecin (SN-
38,4b the active metabolite of irinotecan 6), and 7 were used as
control compounds. Cells were treated with test compounds at
various concentrations under air for 72 h, and cell viability and
proliferation were assessed by the resazurin-based AlamarBlue
assay. The IC50 values for the tested compounds are shown in
Table 1.
It has been reported that substitution on the 12 or 14 position
will result in the loss of activity.6 As expected, 2a and 2b were
much less potent than 1a. However, 3a and 3b were surprisingly
potent, exhibiting potencies comparable to the approved camp-
tothecin analogues, 6 and 7, against the H460 cell line. After these
intriguing results, these two compounds were tested against eight
more human cancer cell lines with the in vitro cytotoxicity assay.
The data listed in Table 1 show the potency of 3a and 3b are
comparable to 6 and 7 across all the cell lines profiled.
It is well established that the camptothecin lactone, the
biologically active form, is in equilibrium with the biologi-
cally inactive ring opened carboxylate.10,11 However, this is not
the case for 14-aminocamptothecins. As shown in Scheme 3, the
lactones of 3a and 3b underwent ring-opening under basic
conditions (NaOH/MeOH, rt, 4 h) to form their corresponding
carboxylates 4a and 4b. Upon acidification, the carboxylates did
not reform the lactones but instead formed the lactams, 5a and
5b, and no parent compounds 3a and 3b were detected. 5a and
5b were tested against the H460 cell line and were found to be
several hundred-fold less active relative to 3a and 3b (Table1).
The stability of 3a and 3b was determined in PBS with and
without 40 mg/mL of human serum albumin (HSA) at 37 °C,
pH 7.4, for up to 180 min. No hydrolysis of the lactones, 3a and
3b, was detected by LC/MS/MS. We conclude that 3a and 3b
Figure 1. Structures of camptothecin, topotecan, irinotecan, SN-38,
9-aminocamptothecin, and 14-chlorocamptothecin.
Figure 2. 1H NMR spectra for 1b and 2b.
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Scheme 1
3b have similar activity against the three pairs of cell lines, while
1a was a substrate of MRP1 and not a substrate of MDR1 and
BCRP. Consistent with the published data, 7 is a substrate of all
three efflux pumps and 6 is a substrate of the BCRP pump.19
14-Aminocamptothecins Show Favorable Interspecies
Bone Marrow Toxicity. Many members of the camptothecin
class have been shown to be highly efficacious in mice only to
show excessive bone marrow toxicity in humans.20 9-Amino-
camptothecin (9) displayed excellent in vivo antitumor activity in
different mice xenografts and was investigated in a number of
clinical trials.21 However, it did not show promising antitumor
activity in patients and the dose limiting toxicity was myelosup-
pression. The maximum tolerated dose in patients was much
lower than was anticipated from mouse studies. A subsequent
study found that bone marrow cells from humans are much more
sensitive to 9 than those from mice.20 The interspecies differ-
ences in the sensitivity of bone marrow cells to 3a and 3b was
assessed by in vitro testing on human and mouse bone marrow-
derived myeloid progenitor colony formation22,23 (Table 4). On
the basis of the IC50 values, 3a and 3b were much less toxic than
the control compounds in both mouse and human bone marrow
cells. The isomeric analogue of 3a, compound 9, was relatively
less toxic to mouse bone marrow cells, having an IC50 of 90 nM
against mouse bone marrow cells, which was about 8-fold more
toxic relative to 3a (710 nM) and 3b (720 nM). When 9 was
tested against human bone marrow cells, its toxicity was drama-
tically increased (over 50-fold), while 3a and 3b showed only a
modest increase (4- and 7-fold shift for 3a and 3b, respectively).
With the clinically approved agents, 7 and particularly 6, the
active metabolite of 8, similar selective toxicity to human bone
marrow progenitor cells was observed.
Scheme 2
Pharmacokinetic Studies: 3a and 3b Showed Significant
Brain Penetration. Compounds 7 and 8 have been approved to
treat central nervous system (CNS) tumors and several other
camptothecin analogues have been tested for treating glioblas-
toma in clinical trials.24 The ability of 3a and 3b to reach the brain
was tested in a mouse pharmacokinetic study. Following oral
administration of 3a and 3b (50 mg/kg IP), peak plasma
concentrations of 0.618 and 0.253 μg/mL, respectively, were
reached at 30 and 60 min post-dose and then declined biexpo-
nentially with half-lives of 250 min for each (Table 5). Peak
concentrations of 3a and 3b in the brain were 0.6 and
0.3 μg/mL, respectively, and declined in parallel to plasma
concentrations with half-lives of 210 and 240 min. Time-
averaged brain to plasma concentration ratios were 101% and
70% for 3a and 3b, respectively (Figure 3, Table 5).
Antitumor Activity of 3a and 3b in Ectopic Xenograft
Models. On the basis of the favorable in vitro data for 3a and 3b,
their antitumor activities in the H460 (NSCLC) human tumor
xenograft model were evaluated. H460 cells (1 ꢀ 106) were
subcutaneously implanted in the flanks of pathogen-free homo-
zygous female nude mice (nu/nu, Charles River Laboratories).
When tumor size reached 100-150 mm3, animals were randomized
to 10 mice per treatment group. All tested compounds were
formulated in 5% DMSO, 5% Tween 80, and 1% CMC in water
and were given orally by gavage. Doses and regimens are listed in
Table 6. The doses of 3a and 3b were chosen on the basis of
preliminary studies to define the MTD of each compound when
administered daily for 5 days. On the basis of weight loss and
behavioral signs, the MTDs of 3a and 3b were determined to be 30
and 40 mg/kg, respectively. Daily oral dosing of 7 was considered an
optimal regimen and 2 mg/kg was used as an MTD.25
are very stable in the presence of physiological concentrations
of HSA.
The cytotoxicity of 3a and 3b on the H460 cell line in the
presence of 40 mg/mL of HSA showed that the activity of 3a was
reduced 5- to 10-fold, while the parent compound, 1a, exhibited a
reduction of more than 77-fold (Table 2). This shift of cytotoxi-
city may be due to the reversible binding of 3a and 3b to HSA. As
expected, the potency of 6 and 7 were only slightly affected by the
presence of HSA because the equilibrium of their lactone and
carboxylate was not affected by HSA.12-14
14-Aminocamptothecins Are Not Substrates for the Clini-
cally Relevant Drug Resistant Pumps, MDR1, MRP1, and
BCRP. One of the main causes of treatment failure in cancer is
the development of drug resistance. Increased efflux of drug by
P-glycoprotein (P-gp, MDR1), multidrug resistance protein
(MRP1), and breast cancer resistance protein (BCRP) limits
the uptake of drug by tumor cells.15a Additionally, these pumps
are responsible for decreasing oral bioavailability and brain
penetration.15b 7 is a substrate of these three major drug resistant
pumps (MDR1, MRP1, and BCRP), while 6 is a substrate of
BCRP and MRP1.19 To assess the liability of 3a and 3b to the
drug resistant pumps, compounds were tested for cytotoxicity in
efflux pump overexpressing cell lines versus the wild type cell
lines from which the overexpressing lines were derived. The three
pairs were MESSA and the MDR1 overexpressing cell line
DX5,16 H69 and the MRP1 overexpressing cell line H69AR,17
and pcDNA and the BCRP overexpressing cell line BCRP
(Table 3).18 The results in Table 4 demonstrate that 3a and
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Scheme 3
Table 2. Effect of HSA on the Cytotoxic Potency of 1a and 3a
and 3ba
IC50 (nM)
(- HSA)
(þ HSA)
IC50 (nM)
95% CI
IC50 (nM)
95% CI
ratio
1a
7
13
59
15
23
12
8-18
40-90
8-21
15-36
6-20
>1000
110
27
>77
2
82-220
19-53
6
2
3a
3b
120
120
93-320
95-300
5
10
a H460 cell line in vitro.
In the H460 xenograft model, both 3a and 3b exhibited
superior antitumor activity compared with 7 (Table 6 and
Figure 4) when administered once daily for five consecutive
days, followed by two days without treatment for two weeks
(QD5 ꢀ 2). Treatment with 3a and 3b yielded 79% and 84%
tumor growth inhibition (TGI) and 15 and 14 days tumor
growth delay (TGD), respectively, with mean maximal body
weight loss of about 4%. In the same model, 7 showed only a 46%
TGI with a maximum body weight loss of 9%.
Both 3a and 3b showed similar antitumor activity in the H460
xenograft model. However, 3b was much easier to formulate
relative to 3a, resulting in a homogeneous suspension for gavage
using the 5% DMSO, 5% Tween 80, and 1% CMC in water
formulation. Limited formulation experimentation with 3a did
not identify an improved formulation (data not shown), so
subsequent efficacy models focused on 3b.
3b was evaluated in the HT29 colon cancer and PC-3 prostate
cancer xenograft models. In the HT29 xenograft model, 3b
exhibited a TGI of 101% and TGD of 30 days as compared to
70% TGI and a TGD of 16 days for 7. In addition, 10 days after
the last dose, 50% of the tumors (5/10) in the 3b treated group
had an objective response (OR) defined as tumors not growing
for at least 10 days after the last dose. This is in contrast to 0%
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ARTICLE
(0/10) OR in the vehicle group and only 10% (1/10) in the 7
treated group.
treatment, body weight of the treated animals recovered rapidly.
In all three models, the antitumor activity of 3b was superior to
7 with daily oral dosing at maximum tolerated doses.
In the PC-3 xenograft model, 3b significantly inhibited tumor
growth as compared to vehicle (p < 0.001) and 7 (p < 0.05,
one-way analysis of variance with Dunnett post-hoc test). TGI of
3b and 7 were 107% and 79% and TGD was 46 and 21 days,
respectively. Furthermore, the OR was 100% (10/10) for 3b and
10% (1/10) for 7. 3b showed mean maximal body weight loss of
7% with no animal exceeding 20% during treatment. Following
Summary. A highly regioselective method has been devel-
oped for synthesizing 14-aminocamptothecins. 3a and 3b dem-
onstrated broad activity against tumor cells in vitro. 3b and the
clinically approved camptothecin analogue, 7, were directly
compared by oral administration in three different human cancer
xenograft models. 3b demonstrated improved antitumor activity
relative to 7 in all three models. Additionally 3b possesses
significant brain penetration, favorable efflux pump properties,
and hematological toxicity profile. On the basis of these results,
3b warrants advancement into clinical development.
Table 3. Drug Resistance Profiling of 3a and 3b on
(a) MDR1, (b) MRP1, and (c) BCRP
(a) MDR1
’ EXPERIMENTAL SECTION
MESSA
IC50 (nM)
DX5
1H and 13C NMR spectra were recorded on a Varian 400 MHz
spectrometer (400 and 75 MHz, respectively) using CDCl3, CD3OD,
or DMSO-d6 as solvents with TMS as an internal standard. HPLC was
performed on an Agilent 1100 series. Mass spectra analysis was
performed on an API 3000 mass spectrometer. Column chromatogra-
phy was performed with silica gel (230-400 mesh). The purity of the
final compounds, 2b, 3a, 3b, 5a, and 5b were determined by LC/MS;
due to the solubility limitation, the purity of 2a could not be analyzed.
The LC traces and the parameters for the LC/MS method are listed in
the Supporting Information, and the purity of the final compounds 3a
and 3b are more than 95% by HPLC. All chemicals were purchased from
Sigma-Aldrich and Fisher Scientific.
14-Nitrocamptothecin (2a). At room temperature (rt), 1a
(2.0 g) was slowly added to a solution of nitric acid (90%, 20 mL)
and the resulting mixture was then stirred overnight. After the reaction
completion, the mixture was poured into ice-water (300 mL) and
filtered under reduced pressure to yield a light-yellow solid. The solid
was then washed with water (50 mL), methanol (50 mL), and ethyl ether
(50 mL). 14-NO2-Camptothecin was obtained (1.0 g) as a light-yellow
powder. 1H NMR (DMSO-d6) δ 8.76 (s, 1H, H-7), 8.16 (d, J = 8.0 Hz,
1H, Ar), 8.05 (d, J = 8.4 Hz, 1H, Ar), 7.88 (t, J = 7.6 Hz, 1H, Ar),
95% CI
IC50 (nM)
95% CI
RRa
1a
7
57
4
40-66
1-10
6-12
44-74
2-6
52
33
22
60
12
30-64
13-57
13-29
27-73
6-22
1
8
2
1
3
6
10
58
4
3a
3b
(b) MRP1
H69
H69AR
IC50 (nM) 95% CI
IC50 (nM)
95% CI
RRa
1a
7
1
6
1-4
>300
>300
250
91
>250
>47
11
1-8
6
22
91
110
7-36
36-110
54-230
69-380
44-120
67-290
3a
3b
1
240
2.2
(c) BCRP
Table 5. Pharmacokinetics of 3a and 3b after a Single Oral
Dose of 50 mg/kg IP to CD-1 Mice
pcDNA
IC50 (nM)
BCRP
95% CI
IC50 (nM)
95% CI
RRa
3a
3b
1a
7
7
10
<1
7
5-9
11
480
220
14
11-18
2
plasma
brain
plasma
brain
7-14
51
>220
2
6
17-400
9-31
5-9
Tmax (min)
30
0.6
90
30
0.6
60
0.3
64
60
0.3
45
3a
3b
6-11
3-7
Cmax (μg/mL)
4
5
1
AUC (μg min/mL)
100
210
3
a RR: resistance ratio.
half-life (min)
250
250
240
Table 4. Human and Mouse CFU-GM: Interspecies in Vitro Hematologic Toxicity Differencea
CFU-GM (IC50)
CFU-GM (IC90)
mouse
human
mouse
human
IC50 (nM)
95% CI
IC50 (nM)
95% CI
ratio (Mo/Hu)
IC90 (nM)
IC90 (nM)
ratio (Mo/Hu)
9
90
130
110
710
620
77-100
1
8
1-2
75
16
85
6
700
700
8
50
88
14
100
4
7
110-150
89-130
7-9
6
1
1-2
800
8
3a
3b
450-970
140-1100
10
110
50-210
72-130
4800
4500
1100
650
6
7
a IC50 and IC90 values derived from assays performed in triplicate.
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7.76 (t, J = 7.4 Hz, 1H, Ar), 6.74 (s, 1H, OH), 5.46 (s, 2H, H-17), 5.31 (s,
2H, H-15), 2.00-2.16 (m, 2H, CH2 of 19-Et), 0.93 (t, J = 7.2 Hz, 3H,
CH3 of 19-Et). 13C NMR (DMSO-d6) δ 8.8, 31.4, 51.2, 65.7, 73.8, 120.9,
127.1, 128.6, 129.17, 129.23, 130.1, 130.8, 131.4, 132.4, 139.3, 141.7,
148.3, 150.4, 156.1, 171.3.
and then connected to a balloon filled with hydrogen. After being stir-
red at room temperature for 3 h, Pd/C was removed by filtration and
the filtrate was then concentrated under reduced pressure. The residue
was purified by flash column purification (eluent: DCM:MeOH = 95:5
(V/V) to yield 460 mg of 3a. 1H NMR (DMSO-d6) δ 8.40 (s, 1H, 7-H),
8.00 (d, J = 8.0 Hz, 1H, Ar), 7.95 (d, J = 8.0 Hz,1H, Ar), 7.74 (t, J = 7.6
Hz,1H, Ar), 7.57 (t, J = 7.4 Hz, 1H, Ar), 7.00 (s, 1H, OH), 6.50 (s, 2H,
NH2) 5.42 (d, J = 17.2 Hz, 1H, H-17), 5.30 (d, J = 16.8 Hz, 1H, H-17),
5.14 (s, 2H, H-15), 1.97-2.12 (m, 2H, CH2 of 19-Et), 0.92 (t, J = 7.4 Hz,
3H, CH3 of 19-Et). 13CNMR (DMSO-d6) δ 8.1, 30.2, 49.4, 65.8, 73.9,
121.8, 122.4, 126.2, 126.4, 128.1, 128.2, 128.7, 128.9, 129.9, 130.0, 136.6,
147.2, 152.8, 154.9, 171.9. MS m/z 364.4 (M þ 1), 727.8 (2M þ 1).
C20H17N3O4 mol wt 363.4.
7-Ethyl-14-aminocamptothecin (3b). 2b (1.0 g) was reduced
with H2 under same conditions as for 3a to produce 3b as a yellow
powder at 73% yield. 1H NMR (CDCl3) δ 8.11 (d, J = 8.4 Hz, 1H, Ar),
8.04 (d, J = 8.0 Hz, 1H, Ar), 7.73 (t, J = 7.6 Hz, 1H, Ar), 7.58 (t, J = 7.6
Hz, 1H, Ar), 6.48 (br, s, 2H, NH2), 5.78(d, J = 16.8 Hz, 1H, H-17), 5.26
(d, J = 16.8 Hz, 1H, H-17), 5.22 (s, 2H, H-15), 4.30 (br, s, 1H, OH), 3.13
(q, J = 7.6 Hz, 2H, CH2 of 7-Et), 2.12-2.24 (m, 1H, CH2 of 19-Et),
1.90-2.00 (m, 1H, CH2 of 19-Et), 1.38 (t, J = 7.6 Hz, 3H, CH3 of 7-Et),
1.08 (t, J = 7.4 Hz, 3H, CH3 of 19-Et). 13C NMR (DMSO-d6) δ 8.1, 13.7,
22.0, 30.2, 48.7, 65.8, 73.9, 121.6, 122.9, 123.6, 124.9, 126.3, 126.7, 128.6,
129.0, 129.4, 136.7, 143.6, 147.8, 152.8, 154.3, 172.0. MS m/z 392.2
(M þ 1), 783.7 (2M þ 1). C22H21N3O4 mol wt 391.4.
7-Ethyl-14-nitrocamptothecin (2b). At 0 °C, HNO3 (1 mL)
was slowly added to a suspension of 1b (1.0 g) in Ac2O (30 mL) with
vigorous stirring. After addition of HNO3, the reaction was stirred for
one hour at 0 °C. Ac2O was then removed under reduced pressure, and
the residue was dissolved in CH2Cl2 and MeOH. The resulting clear
solution was poured into water, and then CH2Cl2 was removed under
reduced pressure and filtered to yield a yellow solid. The solid was then
washed with water and pure product was obtained as yellow powder
(560 mg). The filtrate was concentrated, and the residue was purified by
silica gel column chromatography (MeOH in CH2Cl2 from 0 to 7%) to
give 150 mg more of product, resulting in a total yield of 63.4%. 1H NMR
(DMSO-d6) δ 8.31 (d, J = 8.4 Hz, 1H, Ar), 8.04 (d, J = 8.4 Hz, 1H, Ar),
7.87 (t, J = 7.6 Hz, 1H, Ar), 7.78 (t, J = 7.6 Hz, 1H, Ar), 6.72 (s, 1H, OH),
5.47 (d, J = 2.4 Hz, 2H, H-17), 5.35 (d, J = 6.0 Hz, 2H, H-15), 3.24 (m,
2H, CH2 of 7-Et), 2.00-2.16 (m, 2H, CH2 of 19-Et), 1.30 (t, J = 7.4 Hz,
3H, CH3 of 7-Et), 0.93 (t, J = 7.4 Hz, 3H, CH3 of 19-Et). 13C NMR
(DMSO-d6) δ 8.8, 14.6, 22.9, 31.4, 50.4, 65.7, 73.8, 120.7, 124.6, 127.1,
127.2, 128.9, 129.0, 130.92, 130.95, 139.7, 141.7, 146.4, 148.8, 149.7,
156.0, 171.3. MS m/z 422.3 (M þ 1), 843.4 (2M þ 1). C22H19N3O6
mol wt 421.4.
14-Aminocamptothecin (3a). First, 200 mg of 10% Pd/C was
added to a suspension of 560 mg of 2a in MeOH (100 mL) at room
temperature. Then the mixture was purged with nitrogen three times
5a. At room temperature, NaOH (95 mg) was added to a solution
of 3a (100 mg, 0.265 mmol) in MeOH and the reaction mixture was
stirred at room temperature for one hour. TLC showed that the starting
material disappeared completely. The resulting solution was then
acidified with concentrated HCl to a pH of 2. The cyclization of the
carboxylate 4a to 5a completed within 10 min (monitored by TLC).
After removing the solvent under reduced pressure, the residue was
dissolved in CH2Cl2 and washed with brine. The organic layer was then
dried over MgSO4, filtered, and concentrated under reduced pressure.
After silica gel column chromatography purification (eluent: acetone in
toluene from 20 to 100%), a 32% yield was obtained. 1H NMR (DMSO-
d6) δ 10.67 (s, 1H), 8.54 (s, 1H), 8.17 (d, J = 8.4 Hz, 1H), 8.05 (d, J = 8.4
Hz, 1H), 7.83 (t, J = 7.6 Hz, 1H), 7.65 (t, J = 7.6 Hz, 1H), 6.41 (s, 1H),
5.26 (s, 2H), 5.08 (t, J = 5.2 Hz, 1H), 4.66 (m, 2H), 2.14-2.01 (m, 2H),
0.67 (t, J = 7.4 Hz, 3H). 13C NMR (DMSO-d6) δ 7.9, 30.9, 50.4, 54.2,
119.7, 122.6, 126.9, 128.8, 128.9, 129.2, 130.7, 144.6, 144.8, 148.1, 152.8,
158.9, 177.5. MS m/z 364.2 (M þ 1), 726.4 (2M þ 1). C20H17N3O4,
mol wt 363.4.
5b. 5b was synthesized as described for 5a in 38% yield. 1H NMR
(DMSO-d6) δ 10.63 (s, 1H), 8.19 (t, J = 6.8 Hz, 2H), 7.81 (t, J = 7.6 Hz,
1H), 7.66 (t, J = 7.6 Hz, 1H), 6.41 (s, 1H), 5.28 (s, 2H), 5.08 (t, J = 5.2 Hz,
1H), 4.66 (m, 2H), 3.16 (q, J = 7.6 Hz, 1H), 2.12-2.034 (m, 2H),
Figure 3. Pharmacokinetics of 3a and 3b after a single oral dose of
50 mg/kg IP to CD-1 mice.
Table 6. In Vivo Antitumor Activity in Ectopic Xenograft Modelsa
xenograft model
H460 NSCLC
compd
dosage (mg/kg)
dosing regimen
TGI (%)
TGD (days)
max BW loss (%)
7
2
30
40
QD5 ꢀ 2
QD5 ꢀ 2
QD5 ꢀ 2
46
79
84
5
15
14
9
4
4
3a
3b
HT29 colon cancer
PC-3 prostate cancer
7
2
QD5 ꢀ 4
QD5 ꢀ 4
70
16
30
0
3
3b
40
101
7
2
QD5 ꢀ 2
QD5 ꢀ 2
79
21
45
1
8
3b
40
107
a 1 cycle: QD, 5days on/2days off, oral route of administration (po). TGI: tumor growth inhibition = (1 - ΔT/ΔC) ꢀ 100, where ΔT/ΔC presented the
ratio of the change in mean tumor volume of the treated group and of the control group; TGD: tumor growth delay was calculated as the extra days for
the treated tumor to reach 500 mm3 as compared to the control group.
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resulting in the final drug concentration reported. After drug addition,
the plates were incubated for an additional 72 h at 37 °C, 5% CO2, 95%
air, and 100% relative humidity. At the end of this incubation, the viable
cells were quantified using the AlamarBlue assay. The drug concentra-
tion resulting in growth inhibition of 50% (IC50) was calculated using
Prism software (Irvine, CA).
For cells treated with human serum albumin (HSA), 40 mg/mL of
HSA was added to cells and cocultured with compounds for 72 h.
Ex Vivo Hematological Toxicity CFU-GM Assay. Human
bone marrow cells derived from normal bone marrow were thawed on
the day of experiments. Normal mouse bone marrow was extracted from
both femurs on the day of experiments. The culture was set up in
triplicate at 2 ꢀ 104 cells per culture for both human and mouse pro-
genitor cells. Clonogenic progenitors granulocyte-monocyte (CFU-GM)
lineages were assessed in a semisolid methylcellulose-basedmedium (R&D
Systems) containing recombinant rhSCF (50 ng/mL), rhIL-3 (10 ng/
mL), and rhGM-CSF (10 ng/mL). Clonogenic progenitors of the mouse
erythroid (BFU-E) and myeloid (CFU-GM) lineages were assessed in
a methylcellulose based system (R&D Systems) containing rmSCF
(50 ng/mL), rmIL-3 (10 ng/mL), and rhIL-6 (10 ng/mL). Following
10-12 days in culture, the mouse progenitor derived colonies were
assessed and scored. Following 16-18 days in culture, the human
progenitor derived colonies were assessed and scored.
Brain and Plasma Concentrations of 3a and 3b in CD-1
Mice. CD-1 mice weighing approximately 25 g were dosed intraper-
itoneally with 50 mg/kg of 3a or 3b in a vehicle consisting of 5% DMSO
and 5% Tween 80 in water for injection. Blood from individual mice
(three per time point) for pharmacokinetic analysis were collected by
cardiac puncture into EDTA tubes at different times up to 480 min,
mixed, and centrifuged for 5 min at 13000g to obtain plasma. Immedi-
ately following collection of the blood, individual whole brains from each
animal were excised, washed with distilled water, patted dry, flash frozen
in dry ice/acetone, and then weighed. Plasma and brain samples were
stored at -70 °C until analysis. Plasma samples were treated with five
volumes of ice cold acetonitrile containing 1 μg/mL of propranolol
followed by vortex mixing for 30 s to precipitate proteins. Following
centrifugation for 20 min at 3000g, 5 μL of the supernatant was injected
onto a HPLC/MS/MS triple quadrupole mass spectrometer (Sciex
API3200 QTRAP). A BetaBasic 4, 5 μm HPLC column (2.1 mm ꢀ
50 mm) from Thermo Fisher Scientific (Waltham, MA) was used to
separate the drugs and the internal standard, propranolol. To whole brains,
four volumes of water were added and homogenized in a Precellys-24
(Bertin Technologies, Saint Quentin en Yvelines Cedex, France) for two
cycles of 15 s each. The resulting brain homogenate was then treated with
four volumes of ice cold acetonitrile containing 1 μg/mL of propranolol,
centrifuged for 20 min at 3000g, and 5 μL of the resultant supernatant was
injected onto the HPLC/MS/MS system described above.
Effect of HSA on the Lactone-Lactam Ratios. The kinetics
of conversion and the equilibrium concentrations of the lactone and
lactam forms of the camptothecins 3a, 3b, 5a, and 5b were measured
at pH 7.4 in PBS with and without 40 mg/mL HSA. Stock solutions
(10 mM) of the drugs were diluted to 10 μM and incubated for up to
180 min. Triplicate samples of 50 μL each were immediately treated with
four volumes of ice-cold acetonitrile followed by vortex mixing for 30 s to
precipitate proteins and extract the total lactone and lactam forms of the
drugs. Following centrifugation for 20 min at 3000g, 5 μL of the
supernatant was injected onto the HPLC/MS/MS system as described
above.
Figure 4. Growth of sc implanted human xenografts in nude mice
treated with vehicle, 7, or 3b in (A) H460 NSCLC, (B) HT29 colon
cancer, and (C) PC-3 prostate cancer xenograft models. Doses and
regimens are listed in Table 6. Mean ( SE for group of 10 mice.
1.30 (t, J = 7.6 Hz, 3H), 0.66 (t, J = 7.4 Hz, 3H). 13C NMR (DMSO-d6)
δ 7.9, 13.9, 22.1, 30.9, 38.5, 49.7, 54.1, 76.6, 119.7, 123.2, 123.8,
125.6, 126.9, 127.2, 128.8, 129.5, 129.7, 144.6, 144.8, 148.6, 152.2,
158.9, 177.5. MS m/z 392.2 (M þ 1), 783.7 (2M þ 1). C22H21N3O4
mol wt 391.4.
In Vitro Human Tumor Cell Line Cytotoxicity Assay. In
vitro proliferation assays were conducted as described by Meng et al.26 In
brief, exponentially growing cells were seeded at a density of 4 ꢀ 103 cells
per well in a 96-well plate and incubated at 37 °C in 5% CO2, 95% air,
and 100% relative humidity for 24 h prior to addition of test compounds.
Compounds were solubilized in 100% DMSO at 200 times the desired
final test concentration. At the time of drug addition, compounds were
further diluted to 4 times the desired final concentration with complete
medium. Aliquots of 50 μL of compound at specified concentrations
were added to microtiter wells already containing 150 μL of medium,
In Vivo Human Tumor Xenograft Models and Antitumor
Activity. Specific pathogen-free homozygous female (male for pros-
tate cancer models) nude mice (nu/nu, Charles River Laboratories)
were used. Mice were given food and water ad libitum and housed in
microisolator cages. Four to six week old animals were indentified by
microchips (Locus Technology, Manchester, MD, USA) at the time of
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the experiments. All animal studies were approved by the Institutional
Animal Care and Use Committee at Threshold Pharmaceuticals, Inc.
All cell lines were from the American Type Culture Collection
(ATCC, Rockville, MD, USA). Cells were cultured in the suggested
medium with 10% fetal bovine serum and maintained in a 5% CO2
humidified environment at 37 °C.
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implanted to the flank area of the animals (Table 1). When tumor size
reached 100-150 mm3, mice were randomized into experimental or
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3a, 3b, and 7 were formulated in 5% DMSO, 5% Tween 80, and 1%
CMC in water. All compounds were given orally by gavage, QD ꢀ 5/wk
(5 days on, 2 days off) as one cycle, for a total of 2 or 4 cycles. MTD
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of the change in mean tumor volume of the treated group and of the
control group. TGD was calculated as the extra days for the treated tumor
to reach 500 mm3 as compared to the control group. Animals were culled
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considered statistically significant.
’ ASSOCIATED CONTENT
Supporting Information. 1H NMR, 13C NMR spectra
S
b
and LC/MS of 2a, 2b, 3a, 3b, 5a, and 5b and the purities of 2b,
3a, 3b, 5a, and 5b from reverse phase HPLC analysis. This
material is available free of charge via the Internet at http://pubs.
acs.org.
’ AUTHOR INFORMATION
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Corresponding Author
*Phone: 650-474-8231. Fax: 650-474-0408. E-mail: jduan@
thresholdpharm.com.
’ ACKNOWLEDGMENT
We thank Dr. Emer Clarke of ReachBio, Seattle, WA, for
performing the CFU-GM assays and Zhijun Ye for help with the
NMR spectra of the compounds and Marci Robison for help on
the preparation of the manuscript.
’ ABBREVIATIONS USED
BCRP, breast cancer resistance protein; BW, body weight; CPT,
camptothecin; GM-CFU, granulocyte macrophage colony-form-
ing unit; HSA, Human serum albumin:; IC50, the half maximal
inhibitory concentration; MDR1, multidrug resistance 1; MRP1,
multidrug resistance protein 1; MTD, maximum tolerated dose;
NSCLC, non small cell lung cancer; OR, objective response;
PBS, phosphate buffered saline; SAR, structure activity relation-
ship; TGD, tumor growth delay; TGI, tumor growth inhibition
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ment of oral drug treatment by temporary inhibition of drug transporters
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overview. The Oncologist 2002, 7, 516–530.
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