Stable and orally bio-available pro-drugs of CPS11

Article abstract of DOI:10.1016/j.bmcl.2011.09.026

Stable and orally bio-available pro-drugs of CPS11 were synthesized. They are active on human umbilical vein endothelial cell proliferation assay and tube formation assay. The therapeutic efficacy and safety of 4 as a single agent or combined with Taxol in the treatment of MX-1 human breast cancer xenograft were evaluated. Compound 4 as a single agent failed to produce an anti-tumor activity, while it significantly enhanced antitumor potency of Taxol.

Full text of DOI:10.1016/j.bmcl.2011.09.026

Bioorganic & Medicinal Chemistry Letters 21 (2011) 6242–6244
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Bioorganic & Medicinal Chemistry Letters
journal homepage: www.elsevier.com/locate/bmcl
Stable and orally bio-available pro-drugs of CPS11
Aihong Huo a, Hesheng Zhang b, , Yongbiao Guan c, Guanghuai Zeng b, Yingwei Chen b, Xingwen Li b
⇑
a Tianjin Key Laboratory for Modern Drug Delivery & High-Efficiency, School of Pharmaceutical Science and Technology, Tianjin University, Tianjin 300072, China
b Tianjin Hemay Bio-Tech Co., Ltd, 12 West Keyan Road, Tianjin 300192, China
c Institute of Pharmacology and Toxicology, Academy of Military Medical Sciences, Beijing 100850, China
a r t i c l e i n f o
a b s t r a c t
Article history:
Stable and orally bio-available pro-drugs of CPS11 were synthesized. They are active on human umbilical
vein endothelial cell proliferation assay and tube formation assay. The therapeutic efficacy and safety of 4
as a single agent or combined with Taxol in the treatment of MX-1 human breast cancer xenograft were
evaluated. Compound 4 as a single agent failed to produce an anti-tumor activity, while it significantly
enhanced antitumor potency of Taxol.
Received 15 March 2011
Revised 30 August 2011
Accepted 7 September 2011
Available online 13 September 2011
Ó 2011 Elsevier Ltd. All rights reserved.
Keywords:
CPS11
Pro-drug
Anti-cancer
Synergist
Thalidomide (1) was originally marketed as a sedative during
the 1950s.1–3 It was found to be effective to treat leprosy (ery-
thema nodosum leprosum, ENL). FDA approved thalidomide to
treat ENL in 1998.4 Thalidomide was found effective to inhibit
angiogenesis. When combined with dexamethasone, thalidomide
demonstrated impressive efficacy to treat multiple myeloma
(MM). Nonetheless, thalidomide was reported only limited efficacy
against other cancers, especially solid tumors.
However, CPS11 is unstable during storage. CPS11 decomposes
into thalidomide and formaldehyde even in low temperature as
shown in Scheme 1, making the further development of CPS11
impossible. Thus stable CPS11 pro-drug with proper bioavailability
is needed.
Here we wish to report a set of pro-drugs of CPS11 with good
stability and oral bioavailability. Their activities to inhibit angio-
genesis and their synergistic effects with cell toxin anticancer
agent to inhibit tumor growth are also presented.
N-Hydroxymethylthalidomide (CPS11, 2) repressed NF-jB
while stimulating nuclear factor of activated T cells. Principal com-
ponent analysis of cytokine showed that CPS11 had different col-
lective targeting profiles compared with that of thalidomide.5
Further studies revealed that CPS11 significantly inhibited angio-
genesis in rat aortic ring assay. CPS11 is also a potent inhibitor in
human umbilical vascular endothelial cell (HUVEC) proliferation
tube formation assay. Used as a single agent, CPS11 significantly
inhibited tumor growth by 90% on the human prostate cancer
PC3 xenograft model, while thalidomide did not show efficacy in
the same experiment. In lung cancer metastasis model, CPS11 sig-
nificantly reduced the number of lung metastases by 87%, while
thalidomide did not show efficacy in the same experiment.6
CPS11 is well tolerated (maximum tolerated dose of 100 mg/kg).2
Therefore, CPS11 is an attractive drug candidate for further
development.
The pro-drugs of CPS11 were prepared as illustrated in Scheme
2. Thalidomide reacted with formaldehyde aqueous solution to
form CPS11. When reaction was complete, the mixture was cooled
to room temperature. Some CPS11 crystals were added and the
mixture was cooled with ice bath. The solid form was collected
and dried for next step reaction. The best purity of CPS11 is around
90% and the main impurity is thalidomide. Re-crystallization could
not further improve purity.
CPS11 reacted with picolinoyl chloride, nicotinoyl chloride, or
isonicotinoyl chloride to give 5, 6,7 and 7, respectively. Compounds
5, 6, and 7 could be purified by aqueous work-up and re-crystalli-
zation. These are stable and have proper pharmaceutical property
Ο
Ο
O
O
N
O
N
H
H
N
NH
ΟΗ
Ο
O
⇑
Ο
Corresponding author. Address: Tianjin Hemay Bio-Tech Co., Ltd, 12 West
Keyan Road, Nankai District, Tianjin 300192, China. Tel.: +86 22 87890592 571; fax:
+86 22 87899502 532.
O
2
1
E-mail address: zhanghesheng@hemay.com.cn (H. Zhang).
Scheme 1. Decomposition of CPS11.
0960-894X/$ - see front matter Ó 2011 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmcl.2011.09.026

A. Huo et al. / Bioorg. Med. Chem. Lett. 21 (2011) 6242–6244
6243
Ο
Ο
a
N
O
N
O
NH
N
Ο
1
O
Ο
O
ΟΗ
2
b
Ο
N
Ο
R
O
N
5: pyrid-2-yl
6: pyrid-3-yl
7: pyrid-4-yl
O
Ο
R
O
c
Ο
N
O
HX
N
Ο
O
Ο
HX
N
3:HCl
4:PhSO3H
O
Scheme 2. Synthesis of CPS11 pro-drugs. Reagents and conditions: (a) 30% CH2O aqueous solution, reflux, 1 h, yield: 53.9%; (b) RC(O)Cl, TEA, DCM, rt, overnight, yields: 80–
90%; (c) dilute hydrochloric acid, or PhSO3H, DCM, 30 min.
(results will be published elsewhere). They all are metabolized into
CPS11 in the preliminary pharmacokinetic screening in rats and
meet requirement for further evaluation. Compound 6 was chosen
for efficacy study and salt form screening because its another
metabolite except CPS11 generated in vivo is the endogenous nic-
otinic acid which is not toxic.
The stability of CPS11, 3, and 4 was determined by HPLC. The
purity of CPS11 decreased to 58.9% from 91.6% when stored at
room temperature for 1 month. While after 2 months storage, the
purity of 3 and 4 remained unchanged as their initial purity of
99.3% and 98.9%, respectively. The water solubility of 3 and 4,
determined using UV, is 1.48 and 0.84 mg/mL, respectively. Com-
pounds 3 and 4 are stable more than 8 h in aqueous solution. They
are more stable than compounds reported by Hess.3 After single
intragastric administration to rats, the bioavailability of 4 is
60.7%. When intravenously administered, 4 is rapidly metabolized
into CPS11, indicating that 4 behaves as pro-drug of CPS11 in vivo.
Compounds 5, 6, and 7 inhibit ECV-304 cell growth in a concen-
tration dependent fashion.8 Their IC50s to inhibit the growth of cell
and 4 combined with Taxol treated groups were 33.1% and 74.3%,
respectively, suggesting that 4 could enhance anti-cancer efficacy
of Taxol in this experiment. Compound 4 did not increase the tox-
icity of Taxol. All mice survived and no significant body weight loss
was observed.
In summary, novel pro-drugs of CPS11, 3, and 4 are stable,
water soluble, and oral bio-available. The in vitro anti-angiogenic
efficacy of 6, the free base form of 4, is better than that of thalido-
mide as demonstrated in ECV-304 cell growth assay and HUVEC
tube formation assay. Significant synergic anti-cancer effect of 4
and Taxol is observed on MX-1 human breast cancer xenograft
model. Efficacy of 4 to inhibit cancer metastasis is being evaluated
on lung metastasis model and will be reported on due date. Com-
pound 4 is well tolerated when administered as a single agent or
with Taxol. These results warrant further evaluation of 4 as a novel
anti-cancer agent.
Supplementary data
line ECV-304 were 0.1, 0.12, and 0.18 lM, respectively. The maxi-
mum inhibition rate for thalidomide in the same assay was around
40%.
Supplementary data associated (general procedures and spec-
tral data) with this article can be found, in the online version, at
doi:10.1016/j.bmcl.2011.09.026.
HUVEC proliferation was significantly decreased (88–94%) by 5,
6, and 7 at 195
formation at 195
l
M.9 Thalidomide was found to inhibit 53% tube
References and notes
lM in the same assay.
1. Warfel, N. A.; Lepper, E. R.; Zhang, C. Y.; Figg, W. D.; Dennis, P. A. Cancer Ther.:
Preclinical 2006, 12, 3502.
2. Ng, S. S. W.; Gütschow, M.; Weiss, M.; Hauschildt, S.; Teubert, U.; Hecker, T. K.;
Luzzio, F. A.; Kruger, E. A.; Eger, K.; Figg, W. D. Cancer Res. 2003, 63, 3189.
3. Hess, S.; Akermann, M. A.; Wnendt, S.; Zwingenberger, K.; Eger, K. Bioorg. Med.
Chem. 2001, 9, 1279.
Many studies showed that inhibitors of angiogenesis like thalid-
omide can enhance the anti-tumor efficacy of cell toxin anticancer
agents and Taxol is one of the most effective anti-tumor agents
currently used for the therapy of breast cancer with microtubule
stabilizing properties.10 Anti-cancer efficacy of compound 4 and
the synergic anti-cancer effect of 4 and Taxol were carried out on
MX-1 human breast cancer xenograft model.11 Compound 4 was
administered daily at a dosage of 65 mg/kg ip to mice with estab-
lished tumors from day 7 after inoculation for five consecutive
days. Taxol was administered at a dosage of 5 mg/kg iv on day 7
after inoculation. No significant efficacy on tumor growth in group
treated by 4 was observed. Tumor growth inhibition rates in Taxol
4. Nightingale, S. L. JAMA 1998, 280, 872.
5. Ge, Y.; Montano, I.; Rustici, G.; Freebern, W. J.; Haggerty, C. M.; Cui, W. M.;
Ponciano-Jackson, D.; Chandramouli, G. V. R.; Gardner, E. R.; Figg, W. D.; Abu-
Asab, M.; Tsokos, M.; Jackson, S. H.; Gardner, K. Blood 2006, 108, 4126.
6. Ng, S. S. W.; MacPherson, G. R.; Gütschow, M.; Eger, K.; Figg, W. D. Clin. Cancer
Res. 2004, 10, 4192.
7. 1H NMR of compound 6 (CDCl3, ppm) d 9.2(s, 1H), 8.78(d, 1H, J = 4.0 Hz), 8.29(d,
1H, J = 8.0 Hz), 7.87–7.90(m, 2H), 7.75–7.78(m, 2H), 7.41(dd, 1H, J = 4.0, 8.0 Hz),
6.17(d, 1H, J = 9.6 Hz), 6.09(d, 1H, J = 9.6 Hz), 5.09–5.14(m, 1H), 3.02–3.17(m,
1H), 2.80–2.95(m, 2H), 2.17–2.28(m, 1H); MS (M+H) 394.

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A. Huo et al. / Bioorg. Med. Chem. Lett. 21 (2011) 6242–6244
8. Cell proliferation assay: ECV-304 was cultured for several days in RPMI 1640
containing 10% of bovine serum, collected and suspended in RPMI 1640, placed
into a 96-well cell culture plate at a density of 6000 cells/well and allowed to
attach overnight at 37 °C and 5% CO2. The culture medium were changed to
fresh culture medium containing 1% fetal bovine serum and either the vehicle
(DMSO), thalidomide, compound 5, 6, or 7, then cultured for 72 h under the
to attach overnight at 37 °C and 5% CO2. The culture medium were changed to
fresh culture medium containing either the vehicle (0.5% DMSO), thalidomide,
compound 5, 6, or
7 (0.195 lmol/mL), then cultured for 24 h under the
condition of 5% CO2 and 37 °C. Subsequently, each well was photographed and
counted.
10. Toshiyuki, F.; Mitsuo, T.; Dhar, D. K.; Shuhei, U.; Shoichi, K.; Hiroshi, Y.; Hitoshi,
K.; Naofumi, N. Anticancer Res. 2003, 23, 2405.
condition of 5% CO2 and 37 °C. Subsequently, 20 lL of MTT solution was added
to each well of the 96-well cell culture plate and cultured for another 4 h under
the condition of 5% CO2 and 37 °C. Absorbance was read at 535 nm wavelength.
All experiments were done in duplicate and repeated three times.
11. In vivo effect study: Each mouse was inoculated subcutaneously at the right
flank with the MX-1 tumor fragment of 1–2 mm3. The treatments were started
at day 7 after tumor inoculation when the tumor size reached approximately
200 mm3. Each group consisted of five tumor-bearing mice and treated with
vehicle, compd 4 (65 mg/kg, once a day for five consecutive days), and compd 4
(65 mg/kg, once a day for five consecutive days) combined with Taxol (5 mg/
kg, once at day 7 after tumor inoculation).
9. HUVEC proliferation and tube formation assay: 300 lL of Matrigel was added into
24-well cell plate and allowed to gel at 37 °C and 5% CO2 for 30 min. HUVECs
were suspended in M199 containing 20% of bovine serum and 20 ng/mL bFGF,
and placed into 24-well cell plate at a density of 20,000 cells/well and allowed