Structure-activity relationships study at the 3'-N position of pactilaxel. Part 2: Synthesis and biological evaluation of 3'-N-thiourea- and 3'-N-thiocarbamate-bearing paclitaxel analogues

Article abstract of DOI:10.1016/S0960-894X(00)00242-0

The syntheses and preliminary biological evaluation of 3'-N-thiocarbamate- and 3'-N-thiourea-bearing paclitaxel analogues, 4a-f and 5a-e, are described. 3'-N-thiocarbamates 4a-e were found to be more potent than paclitaxel in both the tubulin polymerization assay and the in vitro cytotoxicity assay. Several derivatives of this class such as 4c, 4d, and 4e also exhibited some in vivo activity.(C) 2000 Elsevier Science Ltd. All rights reserved.

Full text of DOI:10.1016/S0960-894X(00)00242-0

Bioorganic & Medicinal Chemistry Letters 10 (2000) 1327±1331
Structure±Activity Relationships Study at the 30-N Position of
Paclitaxel. Part 2: Synthesis and Biological Evaluation of 30-N-
Thiourea- and 30-N-Thiocarbamate-Bearing Paclitaxel Analogues
May Xue, Byron H. Long,y Craig Fairchild,y Kathy Johnston,y
William C. Rose,y John F. Kadow, Dolatrai M. Vyas and Shu-Hui Chen*{
Bristol-Myers Squibb Research Institute, 5 Research Parkway, PO Box 5100, Wallingford, CT 06492-7660, USA
Received 22 February 2000; accepted 12 April 2000
AbstractÐThe syntheses and preliminary biological evaluation of 30-N-thiocarbamate- and 30-N-thiourea-bearing paclitaxel analo-
gues, 4a±f and 5a±e, are described. 30-N-thiocarbamates 4a±e were found to be more potent than paclitaxel in both the tubulin
polymerization assay and the in vitro cytotoxicity assay. Several derivatives of this class such as 4c, 4d, and 4e also exhibited some
in vivo activity. # 2000 Elsevier Science Ltd. All rights reserved.
Introduction
and relative potency of taxane analogues has stimulated
further e€orts at 30-N modi®cation. As a result of such
e€orts, several cytotoxic 30-N-Boc mimics have been
reported including isosteric 30-N-urea derivatives such as
39a and the isomeric 30-carbamate derivatives.9b In this
report, we describe for the ®rst time, the synthesis and bio-
logical evaluation of two novel series of sulfur containing
paclitaxel side-chain analogues, namely, 30-N-thiocarba-
mates 4 and 30-thiourea derivatives 5 (see Fig. 1).
Paclitaxel, the active ingredient of the anticancer drug
TAXOL1 (Bristol-Myers Squibb Co.), has demonstrated
antitumor activity against a variety of malignancies that
include ovarian, breast, germ cell, lung, and esophageal
cancers.1 3 The taxanes have attracted considerable
attention from the research community due to pacli-
taxel's broad spectrum of clinical activity and its novel
mechanism of action, the promotion of microtubule
assembly and the stabilization of the resulting micro-
tubules.4 Numerous reports have appeared describing
e€orts to identify analogues or formulations that might
have increased bene®ts or advantages over paclitaxel or
docetaxel, the two taxanes that are the active ingredients
of the drugs approved for clinical use.5 7
Chemical Synthesis
The synthetic route employed for the preparation of 30-
free amino derivatives 9a±b and 30-N para-nitrophenyl
carbamates 10a±b, two key precursors for the target
molecules 4 and 5, is shown in Scheme 1. C-13 acylation
of the 7-TES baccatin 610 with b-lactams 7a±b11 was
performed according to the protocol of Holton±
Ojima,12 and provided the desired 30-N protected deri-
vatives 8a±b. Removal of 30-N protecting group was
a€ected by standard hydrogenation leading to the
desired 30-free amines 9a±b in 59±94% yield, respec-
tively. Reaction of these amines with para-nitrophenyl
chloroformate and Hunig's base thus provided the 30-N
carbamates 10a±b in excellent yields.
A number of analogues have incorporated modi®ca-
tions at the side chain 30-N position.5 7 Replacement of
the 30-N-benzamide with a 30-N-tBoc moiety, such as in
docetaxel 2a or 10-acetyl docetaxel 2b, has been shown to
generally result in increased potency in cytoxicity or tubu-
lin polymerization assays when comparing analogues
where other modi®cations remain constant.8 However, the
frequent lack of correlation between antitumor ecacy
*Corresponding author. Tel.: +1-317-276-2076; fax: +1-317-276-
5431; e-mail: chen_shu-hui@lilly.com
yCurrent address: Eli Lilly and Company, Lilly Research Labora-
tories, Lilly Corporate Center, Indianapolis, IN 46285, USA
{Current address: Bristol-Myers Squibb Pharmaceutical Research
Institute, PO Box 4000, Princeton, NJ 08543. USA
Scheme 2 shows the syntheses of six 30-N-thiocarbamates
derivatives 4a±f. These analogues were prepared in good
to excellent yields by reacting a THF solution of 30-p-
nitrophenyl carbamate 10a or 10b with a series of butyl/
0960-894X/00/$ - see front matter # 2000 Elsevier Science Ltd. All rights reserved.
PII: S0960-894X(00)00242-0

1328
M. Xue et al. / Bioorg. Med. Chem. Lett. 10 (2000) 1327±1331
Figure 1.
Scheme 1. Reagents and conditions: (i) LHMDS/THF/7a/0 ꢀC, 87% of 8a; LHMDS/THF/7b/0 ꢀC, 80% of 8b; (ii) 50 Psi H2/Pd(OH)2/C/EtOAc,
94% of 9a (60% conversion), or 59% of 9b; (iii) p-NO2C6H4OC(O)Cl/EtPr2N/CH2/Cl2DMAP/0 ꢀC, 97% of 10a; 93% of 10b.
Scheme 2. Conditions: (i) RSNa/THF/0 ꢀC to rt; (ii) 48% HF/Pyridine/CH3CN/5 ꢀC.
Biological Evaluation
propylthiol sodium salts followed by standard desilyla-
tion. The syntheses of the ®ve 30-N-thiourea derivatives
5a±e were achieved in two steps via acylation of the 30-
amino group in 9a or 9b with benzyl, n-butyl and t-butyl
isothiocyanates followed by desilylation (see Scheme 3).
Every side-chain analogue described in this manuscript
(4 and 5) was characterized by its proton, carbon, and
high-resolution mass spectra.
A total of eleven 30-sulfur containing analogues were
evaluated in a tubulin polymerization assay13 and an in
vitro cytotoxicity assay against human colon cancer cell
lines (HCT-116).14 Analogues exhibiting better potency
than paclitaxel in vitro were further tested in an in vivo
screen against murine M 109 lung carcinoma.15

M. Xue et al. / Bioorg. Med. Chem. Lett. 10 (2000) 1327±1331
1329
Scheme 3. Conditions: (i) RN=C=S/cat. pyridine/C6H6; (ii) 48% HF/Pyridine/CH3CN/5 ꢀC.
As summarized in Table 1, all of six 30-N-thiocarbamate
analogues 4a±f were 2±10 times more potent than
paclitaxel in the tubulin polymerization assay. With the
exception of 4f, analogues 4a±e were found up to 5-fold
more cytotoxic than parent paclitaxel. The lack of
cytotoxicity of 4f was surprising. Three of these analo-
gues, 4c±e, were further evaluated after ip (inter-
peritoneal) administration in an in vivo screen against as
ip-implanted M 109 tumor. All three analogues were
active (T/C>125%). However, these analogues pos-
sessed relatively weaker activities as compared to pacli-
taxel 1 and 30-N-Boc derivative 2b in this experiment.
Remarkably, several 30-N-thiocarbamates (4b, 4d, 4e,
and 4f) exhibited excellent in vitro potency against the
paclitaxel resistant HCT-116 human colon cancer cell
line (see R/S ratios listed in Table 1 for details). The
resistance to paclitaxel in this cell line is due to the
overexpression of p-glycoprotein.
were 5-fold less cytotoxic than paclitaxel. The dis-
crepancy observed between these two bioassays may be
attributed to the eciency of drug transport. On the
other hand, three 30-C phenyl containing analogues,
5a±c, were essentially inactive in both the tubulin and
the cytotoxicity assay. The striking di€erent in vitro
activity observed between 30-N-urea bearing derivative 3
and its isosteric 30-N-thiourea analogues 5a±e clearly
indicates that the carbonyl moiety attached to the
30-amino group is a key structural requirement for bio-
logical activity, presumably due to its involvement in
the intramolecular and/or intermolecular hydrogen
bonding.16
In summary, we have succeeded in the synthesis of two
new series of 30-N-sulfur containing analogues as exem-
pli®ed by structures 417 and 5.18 Our preliminary bio-
logical evaluation of these analogues showed that most
of the 30-N-thiocarbamate carrying analogues described
here demonstrated superior potency in vitro to that dis-
played by paclitaxel, while the 30-N thiourea bearing deri-
vatives prepared to date possessed reduced potency with
respect to paclitaxel in the bioassays tested. The superior
Of the ®ve 30-N-thiourea analogues evaluated, only two
30-C furyl bearing analogues, 5d and 5e, exhibited com-
parable activity to that of paclitaxel in the tubulin poly-
merization assay. Disappointingly, analogues 5d±e
Table 1. In vitro and in vivo activities of side chain analogues 4(a±f) and 5(a±e)
Compound
30-N substituent
30-C
Tubulin ratioa
Cytotoxicity
ratiob
IC50 (nM)
HCT-116 (IC50-R/IC50-S)
ip M-109 T/C
(mg/Kg/inj)c
1
2b
3
C(O)Ph
Ph
Ph
Ph
Ph
1.0
0.66
0.72
0.6
0.3
0.2
0.1
0.2
0.2
12
1.0
0.60
1.0
1.6±6.0 (131)
2.4 (12)
145±218 (60)
177 (32)
n.d.
C(O)OBu-t
C(O)NHBu-t
C(O)SBu-n
C(O)SBu-t
C(O)SBu-n
C(O)SBu-t
C(O)SBu-i
C(O)SPr-i
C(S)NHBn
C(S)NHBu-n
C(S)NHBu-t
C(S)NHBu-n
C(S)NHBu-t
4.0 (46.8)
5.8 (29)
4a
4b
4c
4d
4e
4f
5a
5b
5c
5d
5e
0.88
0.30
0.17
0.30
0.28
14.7
>40
21.8
ꢁ50
4.6
n.d.
n.d.
139 (40)
134 (1.25)
145 (50)
n.d.
n.d.
n.d.
n.d.
n.d.
Ph
0.9 (4.7)
0.3 (21.3)
0.9 (2.2)
0.7 (7.7)
32.5 (1.2)
>87
50.2 (64)
96.7 (31)
10.5 (252)
11.3 (40)
2-Furyl
2-Furyl
2-Furyl
2-Furyl
Ph
Ph
Ph
2-Furyl
2-Furyl
18
19
1.0
1.0
4.9
n.d.
aRatio of analogue relative to paclitaxel (EC0.01). Analogues with ratios <1 being more potent.
bIC50(analogue)/IC50(paclitaxel). Ratios < 1 being more potent. IC50-R: IC50 values of analogues determined in the paclitaxel resistant HCT-116
human colon cancer cell line. IC50-S: IC50 values of analogues measured in the paclitaxel sensitive HCT-116 human colon cancer cell line.
cTreatment given on days 5 and 8 post-tumor implant.

1330
M. Xue et al. / Bioorg. Med. Chem. Lett. 10 (2000) 1327±1331
in vitro potency observed with the 30-thiocarbamate
analogues did not appear to translate into better ecacy
in vivo. It is also important to point out that a number of
30-N-thiocarbamates demonstrated impressive potency
against resistant HCT-116 cell line in vitro. We believe
the results reported herein should further our under-
standing of paclitaxel side chain SAR.
17. General procedure for the preparation of 30-thiocarba-
mate bearing paclitaxel analogues 4a±4f: p-Nitrophenyl car-
bamate 10a/10b (0.25 mmol, 1 equiv) was dissolved in dry
THF (4 mL). To this solution at 0 ꢀC was added a suspension
of RSNa (0.33 mmol, 1.3 equiv.) in THF (1 mL). The reaction
mixture was stirred at rt for 90 min. At this point, the solvent
was removed, the resulting residue was puri®ed with silica gel
chromatography (20±30% EtOAc:Hexanes) to provide the
corresponding 20,7-bisTES protected 30-thiocarbamate pacli-
taxel intermediate. This intermediate (0.24 mmol) was then
dissolved in CH3CN (5 mL), and treated at 0 ꢀC with pyridine
(0.70 mL), followed by 48% HF (2.1 mL). The reaction mix-
ture was kept at 5 ꢀC for 12 h. The reaction mixture was then
diluted with EtOAc (75 mL), and washed with 1 N HCl (Â1),
NaHCO3 saturated solution (Â4) and water. The resulting
organic phase was dried and concd in vacuo. The residue was
chromatographed (40±60% EtOAc:Hexanes) to give the
desired ®nal desilylated 30-thiocarbamate derivative in good to
Acknowledgements
We are grateful to Dr. S.E. Klohr for the measurements
of high resolution mass spectra.
References and Notes
1
excellent overall yield. H NMR of 4a (300 MHz, CDCl3) d
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8.13±8.10 (m, 2H), 7.64±7.26 (m, 8H), 6.26 (m, 3H), 5.65 (d,
J=7.0 Hz, 1H), 5.60 (m, 1H), 4.93 (d, J=8.7 Hz, 1H), 4.68
(m, 1H), 4.40 (m, 1H), 4.23 (AB q, J=8.4 Hz, 2H), 3.79 (d,
J=6.9 Hz, 1H), 3.48 (d, J=5.0 Hz, 1H), 2.80±0.74 (m, 31H,
1
incl. singlets at 2.36, 2.24, 1.83, 1.68, 1.26, 1.15, 3H each). H
NMR of 4b (300 MHz, CDCl3) d 8.11±8.08 (m, 2H), 7.60±7.32
(m, 8H), 6.24 (m, 3H), 5.65 (d, J=7.0 Hz, 1H), 5.54 (d, J=8.7
Hz, 1H), 4.93 (d, J=8.0 Hz, 1H), 4.65 (s, 1H), 4.40 (m, 1H),
4.22 (AB q, J=8.5 Hz, 2H), 3.79 (d, J=7.0 Hz, 1H), 3.52 (d,
J=3.9 Hz, 1H), 2.60±1.14 (m, 31H, incl. singlets at 2.36, 2.23,
1
1.83, 1.67, 1.26, 1.14, 3H each, 1.34, 9H). H NMR of 4c (300
MHz, CDCl3) d 8.12±8.09 (m, 2H), 7.59±7.45 (m, 4H), 6.36±
6.21 (m, 4H), 5.64 (d, J=6.9 Hz, 2H), 4.93 (d, J=8.4 Hz, 1H),
4.73 (d, J=3.2 Hz, 1H), 4.40 (m, 1H), 4.21 (AB q, J=8.3 Hz,
2H), 3.80 (d, J=6.8 Hz, 1H), 3.68 (d, J=5.6 Hz, 1H), 2.76±
1.13 (m, 28H, incl. singlets at 2.38, 2.23, 1.86, 1.66, 1.24, 1.13,
1
3H each), 0.76 (t, J=7.1 Hz, 3H). H NMR of 4d (300 MHz,
CDCl3) d 8.11±8.08 (m, 2H), 7.59±7.40 (m, 4H), 6.37±6.17 (m,
4H), 5.64 (m, 2H), 4.94 (d, J=8.3 Hz, 1H), 4.71 (m, 1H), 4.40
(AB q, J=8.4 Hz, 2H), 3.80 (d, J=7.0 Hz, 1H), 3.62 (d,
J=5.5 Hz, 1H), 2.62±1.13 (m, 31H, incl. singlets at 2.39, 2.23,
1
1.87, 1.66, 1.24, 1.14, 3H each, 1.35, 9H). H NMR of 4e (300
8. Georg, G. I.; Boge, T. C.; Cheruvallath, Z. S.; Harriman,
G. C. B.; Hepperle, M.; Park, H.; Himes, R. H. Bioorg. Med.
Chem. Lett. 1994, 4, 335.
MHz, CDCl3) d 8.13±8.10 (m, 2H), 7.61±7.42 (m, 4H), 6.39±
6.15 (m, 4H), 5.66 (d, J=7.1 Hz, 2H), 4.94 (d, J=7.9 Hz, 1H),
4.74 (d, J=2.1 Hz, 1H), 4.41 (dd, J=6.7 Hz, J0=10.8 Hz, 1H),
4.23 (AB q, J=8.4 Hz, 2H), 3.81 (d, J=7.1 Hz, 1H), 2.80±0.78
(m, 25H, incl. singlets at 2.38, 2.24, 1.88, 1.68, 1.25, 1.15, 3H
9. (a) Holton, R. A.; Nadizadeh, H.; Rengan, K.; Tao, C.
PCT Patent: WO 9421251. (b) Chen, S.H.; Xue, M.; Huang,
S.; Long, B. H.; Fairchild, C. A.; Rose, W. C.; Kadow, J. F.;
Vyas, D. M. Bioorg. Med. Chem. Lett. 1997, 7, 3057.
10. Denis, J.-N.; Greene, A.; Guenard, D.; Gueritte-Voelegein,
F.; Mangatal, L.; Potier, P. J. Am. Chem. Soc. 1988, 110, 5917.
11. For a reference on N-Cbz protected b-lactam, see: Mar-
ing, C. J.; Grampovnik, D. J.; Yeung, C. M.; Klein, L. L.; Li,
L.; Thomas, S. A.; Plattner, J. J. Bioorg. Med. Chem. Lett.
1994, 4, 1429.
1
each), 0.82 and 0.80, (d, J=6.7 Hz, 3H each). H NMR of 4f
(300 MHz, CDCl3) d 8.10±8.07 (m, 2H), 7.60±7.38 (m, 4H),
6.35±6.20 (m, 4H), 5.62 (d, J=7.0 Hz, 1H), 4.90 (d, J=8.1 Hz,
1H), 4.71 (s, 1H), 4.38 (m, 1H), 4.20 (AB q, J=8.4 Hz, 2H),
3.78 (d, J=7.0 Hz,1H), 3.66 (s, 1H), 3.45 (m, 1H), 2.60±1.07
(m, 28H, incl. singlets at 2.36, 2.20, 1.84, 1.64, 1.22, 1.10, 3H
each, doublets (J=6.8 Hz, 3H each) at 1.16, 1.09).
1
18. Proton NMR spectra of 3-thiourea derivatives 5a±5e: H
12. Holton, R. A. U.S. Patent 5, 015, 744 1991. (b) Holton,
R. A. U.S. Patent 5, 136, 060 1992. (c) Ojima, I.; Habus, I.;
Zhao, M.; Zucco, M.; Park, Y. H.; Sun, C.-M.; Brigaud, T.
Tetrahedron 1992, 48, 6985.
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Med. Chem. 1991, 34, 1176.
14. Scudiero, D. A.; Shoemaker, R. H.; Paull, K. D.; Monks,
A.; Tierney, S.; Nofziger, T. H.; Currents, M. J.; Seni€, D.;
Boyd, M. R. Cancer Res. 1988, 48, 4827.
NMR of 5a (300 MHz, CDCl3) d 8.05±8.02 (m, 2H), 7.55±7.19
(m, 13H), 6.87 (bs, 2H), 6.23±6.18 (m, 3H), 5.60 (d, J=7.1 Hz,
1H), 4.84 (d, J=9.4 Hz, 1H), 4.72 (s, 1H), 4.49 (m, 2H), 4.32
(m, 1H), 4.18 (AB q, J=9.4 Hz, 2H), 3.72 (d, J=7.0 Hz, 1H),
2.62±1.07 (m, 22H, incl. singlets at 2.35, 2.18, 1.75, 1.63, 1.17,
1.08, 3H each). 1H NMR of 5b (300 MHz, CDCl3) d 8.07±8.04
(m, 2H), 7.60±7.30 (m, 8H), 6.66 (bs, 1H), 6.26 (m, 3H), 5.63
(d, J=7.0 Hz, 1H), 4.90 (d, J=8.4 Hz, 1H), 4.80 (s, 1H), 4.36
(m, 1H), 4.20 (AB q, J=8.4 Hz, 2H), 3.84 (m, 1H), 3.76 (d,
J=6.9 Hz, 1H), 3.13 (bs, 2H), 2.61±1.11 (m, 28H, incl. singlets
at 2.35, 2.21, 1.80, 1.66, 1.22, 1.12, 3H each), 0.86 (t, J=7.2
15. Rose, W. C. Anti-Cancer Drugs 1992, 3, 311.
16. For a thoughful discussion of the involvements of the side
chain polar groups in hydrogen bonding, see: Guenard, D.;
Gueritte-Voegelein, F.; Potier, P. Acc. Chem. Res. 1993, 26,
160, and references cited therein. Also see: Gao, Q.; Wei, J.
M.; Chen, S. H. Pharma. Res. 1995, 12, 337.
1
Hz, 3H). H NMR of 5c (300 MHz, CDCl3) d 8.09±8.06 (m,
2H), 7.58±7.33 (m, 8H), 6.58 (d, J=9.1 Hz, 1H), 6.37±6.26 (m,
3H), 6.18 (s, 1H), 5.64 (d, J=7.1 Hz, 1H), 4.91 (d, J=7.8 Hz,
1H), 4.85 (s, 1H), 4.39 (m, 1H), 4.22 (AB q, J=8.5 Hz, 2H),

M. Xue et al. / Bioorg. Med. Chem. Lett. 10 (2000) 1327±1331
1331
3.78 (d, J=7.1 Hz, 1H), 3.48 (s, 1H), 2.60±1.13 (m, 31H, incl.
singlets at 2.39, 2.21, 1.76, 1.68, 1.25, 1.13, 3H each, 1.39, 9H).
1H NMR of 5d (300 MHz, CDCl3) d 8.08±8.05 (m, 2H), 7.58±
7.41 (m, 4H), 6.45±6.25 (m, 4H), 6.17 (d, J=9.4 Hz, 1H), 6.00
(bs, 1H), 5.64 (d, J=7.1 Hz, 1H), 4.92 (d, J=9.2 Hz, 1H), 4.83
(d, J=2.1 Hz, 1H), 4.40 (m, 1H), 4.22 (AB q, J=8.4 Hz, 2H),
3.79 (d, J=7.0 Hz, 1H), 3.33 (d, J=4.3 Hz, 1H), 3.26 (bs, 1H),
2.60±0.87 (m, 31H incl. singlets at 2.43, 1.85, 1.67, 1.55, 1.23,
1
1.12, 3H each, triplet at 0.90, 3H). H NMR of 5e (300 MHz,
CDCl3) d 8.07±8.04 (m, 2H), 7.56±7.41 (m, 4H), 6.45±6.21 (m,
6H), 5.63 (d, J=7.1 Hz, 1H), 4.92 (d, J=7.9 Hz, 1H), 4.85 (s,
1H), 4.40 (m, 1H), 4.21 (AB q, J=8.5 Hz, 2H), 3.79 (d, J=7.1
Hz, 1H), 3.42 (bs, 1H), 2.60±1.11 (m, 31H, incl. singlets at 2.43,
2.22, 1.83, 1.66, 1.23, 1.11, 3H each, 1.39, 9H).