U.S. patent application number 16/710711 was filed with the patent office on 2020-11-19 for treatment or prophylaxis of proliferative conditions.
The applicant listed for this patent is University Court of the University of Dundee. Invention is credited to Steven Albert Everett, Saraj Ulhaq.
Application Number | 20200360521 16/710711 |
Document ID | / |
Family ID | 1000004991516 |
Filed Date | 2020-11-19 |
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United States Patent
Application |
20200360521 |
Kind Code |
A1 |
Everett; Steven Albert ; et
al. |
November 19, 2020 |
TREATMENT OR PROPHYLAXIS OF PROLIFERATIVE CONDITIONS
Abstract
The invention relates to novel compounds for use in the
treatment or prophylaxis of cancers and other proliferative
conditions that are for example characterized by cells that express
cytochrome P450 1B1 (CYP1B1) and allelic variants thereof. The
invention also provides pharmaceutical compositions comprising one
or more such compounds for use in medical therapy, for example in
the treatment of prophylaxis of cancers or other proliferative
conditions, as well as methods for treating cancers or other
conditions in human or non-human animal patients. The invention
also provides methods for identifying novel compounds for use in
the treatment of prophylaxis of cancers and other proliferative
conditions that are for example characterized by cells that express
CYP1 B1 and allelic variants thereof. The invention also provides a
method for determining the efficacy of a compound of the invention
in treating cancer.
Inventors: |
Everett; Steven Albert;
(Menlo Park, CA) ; Ulhaq; Saraj; (Dundee,
GB) |
|
Applicant: |
Name |
City |
State |
Country |
Type |
University Court of the University of Dundee |
Dundee |
|
GB |
|
|
Family ID: |
1000004991516 |
Appl. No.: |
16/710711 |
Filed: |
December 11, 2019 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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15924520 |
Mar 19, 2018 |
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16710711 |
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15425758 |
Feb 6, 2017 |
9919060 |
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15924520 |
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14485122 |
Sep 12, 2014 |
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15425758 |
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13318321 |
Apr 10, 2012 |
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PCT/GB2010/000860 |
Apr 30, 2010 |
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14485122 |
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61174884 |
May 1, 2009 |
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Current U.S.
Class: |
1/1 |
Current CPC
Class: |
A61K 31/381 20130101;
C07D 407/12 20130101; C07D 307/80 20130101; C07D 491/22 20130101;
A61K 47/545 20170801; A61K 31/4184 20130101; C07D 311/16 20130101;
A61K 31/52 20130101; A61K 31/352 20130101; C07D 409/12 20130101;
A61K 31/513 20130101; A61K 31/428 20130101; A61K 31/343 20130101;
C07D 405/04 20130101; C07D 405/12 20130101; A61K 31/664 20130101;
A61K 31/665 20130101; C07D 487/04 20130101; A61K 31/37 20130101;
C07D 417/12 20130101; C07D 311/18 20130101; C07D 405/14
20130101 |
International
Class: |
A61K 47/54 20060101
A61K047/54; A61K 31/343 20060101 A61K031/343; A61K 31/352 20060101
A61K031/352; A61K 31/4184 20060101 A61K031/4184; A61K 31/428
20060101 A61K031/428; A61K 31/664 20060101 A61K031/664; C07D 307/80
20060101 C07D307/80; C07D 311/16 20060101 C07D311/16; C07D 311/18
20060101 C07D311/18; C07D 405/04 20060101 C07D405/04; C07D 405/12
20060101 C07D405/12; C07D 405/14 20060101 C07D405/14; C07D 407/12
20060101 C07D407/12; C07D 409/12 20060101 C07D409/12; C07D 417/12
20060101 C07D417/12; C07D 487/04 20060101 C07D487/04; C07D 491/22
20060101 C07D491/22; A61K 31/37 20060101 A61K031/37; A61K 31/381
20060101 A61K031/381; A61K 31/513 20060101 A61K031/513; A61K 31/52
20060101 A61K031/52; A61K 31/665 20060101 A61K031/665 |
Foreign Application Data
Date |
Code |
Application Number |
May 1, 2009 |
GB |
0907551.6 |
Claims
1. A compound of formula (I): ##STR00115## (wherein: X.sup.1 is
such that --X.sup.1--X.sup.2 is --O--X.sup.2, --S--X.sup.2,
--SO.sub.2--O--X.sup.2, --SO.sub.2NZ.sup.10--X.sup.2, conjugated
alkenemethyloxy or conjugated alkenemethylthio, conjugated
alkenemethylSO.sub.2--O, conjugated alkenemethyl-SO.sub.2NZ.sup.10
or of the formula: ##STR00116## --X.sup.2 is absent or is such that
X.sup.1--X.sup.2-Effector is one of ##STR00117## each n and m is
independently 0 or 1; p is 0, 1 or 2; X.sup.3 is oxygen or sulfur
and additionally, when m=0, may be SO.sub.2--O, SO.sub.2NZ.sup.10,
conjugated alkenemethyloxy, conjugated alkenemethylthio, conjugated
alkenemethyl-SO.sub.2--O or conjugated
alkenemethyl-SO.sub.2NZ.sup.10 each of Y.sup.1, Y.sup.2 and Y.sup.3
is independently carbon or nitrogen, wherein if Y.sup.1 is
nitrogen, Z.sup.1 is absent, if Y.sup.2 is nitrogen, Z.sup.3 is
absent and if Y.sup.3 is nitrogen, Z.sup.5 is absent; Y.sup.4 is an
oxygen, carbon or nitrogen atom, sulfoxide or sulfone; --Y.sup.5--
is either (i) a single bond, (ii) .dbd.CH--, wherein the double
bond = in .dbd.CH-- is connected to Y.sup.4, or (iii) --CH.sub.2--
or --CH.sub.2CH.sub.2--, or one of (ii) to (iii) wherein the
hydrogen atom in (ii) is or one or more hydrogen atoms in (iii) are
replaced with a substituent Z.sup.11, wherein Z.sup.11 is selected
independently from alkyl, alkenyl, alkynyl, aryl, aralkyl,
alkyloxy, alkenyloxy, alkynyloxy, aryloxy, aralkyloxy, alkylthioxy,
alkenylthioxy, alkynylthioxy, arylthioxy, aralkylthioxy, amino,
hydroxy, thio, halo, carboxy, formyl, nitro and cyano; each of
Z.sup.1--Z.sup.4, where present, are independently selected from
hydrogen, alkyl, alkenyl, alkynyl, aryl, aralkyl, alkyloxy,
alkenyloxy, alkynyloxy, aryloxy, aralkyloxy, alkylthioxy,
alkenylthioxy, alkynylthioxy, arylthioxy, aralkylthioxy, amino,
hydroxy, thio, halo, carboxy, formyl, nitro and cyano; and Z.sup.5,
where present, is independently selected from hydrogen alkyl,
alkenyl, alkynyl, aryl, aralkyl, alkyloxy, alkenyloxy, alkynyloxy,
aryloxy, aralkyloxy, alkylthioxy, alkenylthioxy, alkynylthioxy,
arylthioxy, aralkylthioxy, amino, hydroxy, thio, carboxy, formyl,
nitro and cyano, or one of Z.sup.2 & Z.sup.3, Z.sup.3 &
Z.sup.4 and Z.sup.4 and Z.sup.5 together with the atoms to which
they are connected form an aromatic ring fused to the remainder of
the compound, provided that at least one of Z.sup.1, Z.sup.2 and
Z.sup.4 is hydrogen; Z.sup.6 is selected from hydrogen, alkyl,
alkenyl, alkynyl, aryl and aralkyl; none, one or two of Y.sup.6 may
be nitrogen atoms with the remainder being carbon atoms; each
Z.sup.7 is independently hydrogen, alkyl or aryl; each Z.sup.8 is
independently selected from hydrogen, an electron withdrawing
group, unsubstituted C.sub.1-C.sub.6 alkyl, substituted
C.sub.1-C.sub.6 alkyl, unsubstituted C.sub.1-C.sub.6 alkoxy, and
substituted C.sub.1-C.sub.6 alkoxy where the substituted alkyl or
alkoxy are substituted with one or more groups selected from ether,
amino, mono- or di-substituted amino, cyclic C.sub.1-C.sub.5
alkylamino, imidazolyl, C.sub.1-C.sub.6 alkylpiperazinyl,
morpholino, thiol, thioether, tetrazole, carboxylic acid, ester,
amido, mono- or di-substituted amido, N-connected amide,
N-connected sulfonamide, sulfoxy, sulfonate, sulfonyl, sulfoxy,
sulfinate, sufinyl, phosphonooxy, phosphate and sulfonamide; each
Z.sup.9 is independently oxygen or sulfur; Z.sup.10 is hydrogen or
alkyl, for example a C.sub.1-4 alkyl; Effector is a molecule having
a pharmacological or diagnostic function), or a pharmaceutically
acceptable salt, ester, amide or solvate thereof.
Description
FIELD
[0001] The present invention relates to novel compounds for use in
the treatment or prophylaxis of cancers and other proliferative
conditions that are for example characterized by cells that express
cytochrome P450 1B1 (CYP1B1) and allelic variants thereof. The
present invention also provides pharmaceutical compositions
comprising one or more such compounds for use in medical therapy,
for example in the treatment of prophylaxis of cancers or other
proliferative conditions, as well as methods for treating cancers
or other conditions in human or non-human animal patients. The
present invention also provides methods for identifying novel
compounds for use in the treatment of prophylaxis of cancers and
other proliferative conditions that are for example characterized
by cells that express CYP1B1 and allelic variants thereof. The
present invention also provides a method for determining the
efficacy of a compound of the invention in treating cancer.
BACKGROUND
[0002] CYP1B1 is a member of the dioxin-inducible CYP1 gene family
which also includes CYP1A1 and CYP1A2 as described by Sutter et al.
(J Biol. Chem., May 6; 269(18):13092-9, 1994). CYP1B1 is a
heme-thiolate mono-oxygenase enzyme that is capable of metabolizing
and activating a variety of substrates including steroids,
xenobiotics, drugs and/or prodrugs. CYP1B1 protein is expressed to
a high frequency in a wide range of primary and metastatic human
cancers of different histogenic types and is not expressed or at
negligible levels in normal tissue. (see, e.g.: McFadyen M C,
Melvin W T and Murray G I, "Cytochrome P450 Enzymes: Novel Options
for Cancer Therapeutics", Mol Cancer Ther., 3(3): 363-71, 2004;
McFadyen M C and Murray G I, "Cytochrome P450 1B1: a Novel
Anticancer Therapeutic Target", Future Oncol., 1(2): 259-63, 2005;
Sissung T M, Price D K, Sparreboom A and Figg W D,
"Pharmacogenetics and Regulation of Human Cytochrome P450 1B1:
Implications in Hormone-Mediated Tumor Metabolism and a Novel
Target for Therapeutic Intervention", Mol. Cancer Res.,
4(3):135-50, 2006).
[0003] More specifically, CYP1B1 has been shown to be expressed in
bladder, brain, breast, colon, head and neck, kidney, lung, liver,
ovarian, prostate and skin cancers, without being expressed in the
corresponding normal tissue. For example, Barnett, et al, in Clin.
Cancer Res., 13(12): 3559-67, 2007, reported that CYP1B1 was
over-expressed in glial tumours, including glioblastomas,
anaplastic astrocytomas, oligodendrogliomas and anaplastic
oligodendrogliomas, but not unaffected brain tissue; Carnell, et
al., in Int. J. Radiat. Oncol. Biol. Phys., 58(2): 500-9, 2004,
reported that CYP1B1 was over-expressed in prostate
adenonocarcinomas, but not in matched normal prostate tissue;
Carnell, et al., 2004 (ibid.) also showed that CYP1B1 is expressed
in (n=22, 100%) of bladder carcinomas; Downie, et al., in Clin.
Cancer Res., 11(20): 7369-75, 2005 and McFadyen, et al., in Br. J.
Cancer, 85(2): 242-6, 2001, reported increased expression of CYP1B1
in primary and metastatic ovarian cancer, but not in normal ovary
tissue; and Gibson, et al., in Mol. Cancer Ther., 2(6): 527-34,
2003, and Kumarakulasingham, et al., in Clin. Cancer Res., 11(10):
3758-65, 2005, reported that CYP1B1 was over-expressed in colon
adenocarcionomas as compared to matched normal tissue.
[0004] Several studies have shown that CYP1B1 is over-expressed in
breast cancer as compared to matched normal tissue (see, e.g.:
Murray G I, Taylor M C, McFadyen M C, McKay J A, Greenlee W F,
Burke M D and Melvin W T, "Tumor-Specific Expression of Cytochrome
P450 CYP1B1", Cancer Res., 57(14): 3026-31, 1997; Haas S, Pierl C,
Harth V, Pesch B, Rabstein S, Bruning T, Ko Y, Hamann U,
Justenhoven C, Brauch H and Fischer H P, "Expression of Xenobiotic
and Steroid Hormone Metabolizing Enzymes in Human Breast
Carcinomas". Int. J. Cancer, 119(8): 1785-91, 2006; McKay J A,
Murray G I, Ah-See A K, Greenlee W F, Marcus C B, Burke M D and
Melvin W T, "Differential Expression of CYP1A1 and CYP1B1 in Human
Breast Cancer", Biochem. Soc. Trans., 24(2): 327S, 1996).
[0005] Everett, et al., in J. Clin. Oncology, 25: 18S, 2007,
reported that CYP1B1 was over-expressed in malignant melanoma and
disseminated disease but not in normal skin. Chang, et al., in
Toxico. Sci., 71(1): 11-9, 2003, reported that CYP1B1 protein is
not present in normal liver but Everett, et al., 2007 (ibid.)
confirmed CYP1B1 over-expression in melanoma stage IV metastasis to
the liver but not in the adjacent normal liver tissue.
[0006] Greer, et al., in Proc. Am. Assoc. Cancer Res., 45: 3701,
2004, reported that CYP1B1 was over-expressed during the malignant
progression of head and neck squamous cell carcinoma but not in
normal epithelium.
[0007] McFadyen, et al., in Br. J. Cancer, 91(5): 966-71, 2004,
detected CYP1B1 in renal carcinomas but not in corresponding normal
tissue.
[0008] Murray, et al., 2004 (ibid.) used immunohistochemistry to
show over-expression of CYP1B1 in lung cancer cells as compared to
normal lung tissue. Su, et al., in Anti-Cancer Res., 2, 509-15,
2009, used immunohistochemistry to show over-expression of CYP1B1
in advanced stage IV non-small cell lung cancer compared to earlier
stages of the disease.
[0009] It is evident from the numerous disclosures cited above that
CYP1B1 expression is characteristic of a range of different cancers
and other proliferative conditions, and that CYP1B1 expression may
be used to define such a range of cancers and other conditions. As
normal (non-cancerous) cells do not express significant levels of
CYP1B1, it may also be reasonably expected that compounds that
exhibit cytotoxicity in cells expressing CYP1B1, but are
substantially non-cytotoxic in normal cells, would have utility as
targeted anti-cancer agents in cancers characterized by CYP1B1
expression. By "targeted" is meant that such compounds could be
delivered systemically and would only be activated in the presence
of cancerous cells expressing CYP1B1, remaining substantially
non-toxic to the rest of the body.
[0010] Furthermore, a number of cytochrome P450 enzymes are known
to metabolise and detoxify a variety of anticancer drugs. McFadyen,
et al. n (Biochem Pharmacol. 2001, Jul. 15; 62(2): 207-12)
demonstrated a significant decrease in the sensitivity of docetaxel
in cells expressing CYP1B1 as compared with non-CYP1B1 expressing
cells. This finding indicates that the presence of CYP1B1 in cells
may decrease their sensitivity to some cytotoxic drugs.
CYP1B1-activated prodrugs may therefore be useful for the treatment
of cancers whose drug resistance is mediated by CYP1B1.
[0011] Furthermore, the CYP1B1 gene is highly polymorphic in cancer
and several single nucleotide polymorphisms contained within the
CYP1B1 gene have been identified that alter the expression and/or
activity of the encoded protein. Of these, the CYP1B1*3
(4326C>G; L432V) allele has been characterized by both increased
expression and enzyme kinetics of CYP1B1 toward several substrates
as described by Sissung, et al. in Mol Cancer Ther., 7(1): 19-26,
2008 and references quoted therein. This finding indicates that not
only CYP1B1 but the allelic variants of the enzyme may also
contribute to prodrug activation and cancer targeting.
[0012] Prodrugs have been investigated as a means to lower the
unwanted toxicity or some other negative attribute of a drug
without loss of efficacy. A prodrug is a drug that has been
chemically modified to render it inactive but that, subsequent to
administration, is metabolized or otherwise converted to an active
form of the drug in the body. The over-expression of CYP1B1 in
primary tumours and metastatic disease compared to normal tissue
offers a tremendous opportunity for the development of
CYP1B1-activated prodrugs for targeted cancer therapy as reviewed
by McFadyen et al., Mol Cancer Ther., 3(3), 363-71, 2004. Indeed,
the discovery and development of CYP1B1-activated prodrugs for
targeted cancer therapy is likely to offer significant
pharmacological advantages over existing non-targeted cytochrome
P450-activated prodrugs used clinically such as the prodrug
alkylating agents cyclophosphamide, ifosfamide, dacarbazine,
procarbazine which are activated by cytochrome P450s expressed in
normal tissue as reviewed by Patterson L H and Murray G I in Curr
Pharm Des., 8(15): 1335-47, 2002.
[0013] The human cytochrome P450 family contains 57 active
isozymes, which function in normal metabolism, influence drug
pharmacokinetics and effect negative outcomes in patients through
drug-drug interactions. The cytochrome P450 isoenzymes metabolize
approximately two thirds of known drugs in humans, with 80% of this
attributable to five isozymes, namely CYP1A2, CYP2C9, CYP2C19,
CYP2D6, and CYP3A4 as described in Ortiz de Montellano, PR (ed.)
Cytochrome P450: structure, mechanism, and biochemistry, Kluwer
Academic/Plenum Publishers, New York, 2005.
[0014] Among the genes discovered by intiatives in the human genome
project are CYP2R1, CYP2W1, CYP2S1, CYP2S1, CYP2U1 but the
function, polymorphism and regulation of these genes are still to
be fully elucidated as reviewed by Ingelman-Sundberg, M., Toxicol.
Appl. Pharmacol., 207, 52-6, 2005. In addition to CYP1B1 a number
of these cytochrome P450 oxidoreductases are extrahepatic and
over-expressed in cancer. Several cytochrome P450s including
CYP1B1, CYP2A/2B, CYP2F1, CYP2R1, CYP2U1, CYP3A5, CYP3A7, CYP4Z1,
CYP26A1, and CYP 51 are present at a significantly higher level of
intensity than in normal ovary as determined by
immunohistochemistry and light microscopy, as described by Downie
et al., Clin. Cancer Res., 11(20): 7369-75, 2005. Furthermore,
using similar methods of detection in primary colorectal cancer,
several cytochrome P450s, including CYP1B1, CYP2S1, CYP2U1, CYP3A5,
and CYP51, are frequently over-expressed compared to normal colon
as descried by Kumarakulasingham et al, Clin. Cancer Res., 11(10):
3758-65, 2005. In the same study several cytochrome P450s,
including CYP1B1, CYP2A/2B, CYP2F1, CYP4V2, and CYP39, correlated
with their presence in the primary tumour. CYP2W1 has also been
shown to be over-expressed in colorectal cancer according to Elder
et al, Eur. J. Cancer, 45(4): 705-12. CYP4Z1 is over-expressed in
breast carcinoma is a gene associated with non-small cell lung
cancer promotion and progression as described by Reiger et al.,
Cancer Res., 64(7): 2357-64, 2004 and Bankovic et al., Lung Cancer,
67(2): 151-9, 2010, respectively.
[0015] A major challenge in the field is elucidation of the
function of human cytochrome P450s of so-called `orphan` status
with unknown substrate specificity as reviewed by Strak K and
Guengerich F P in Drug Metab. Rev., 39(2-3): 627-37, 2007. A number
of substrates are known for CYP1B1 few of which are specifically
metabolised by the enzyme, for example 7-ethoxyresorufin undergoes
oxidative de-ethylation when activated by all members of the CYP1
family, including CYP1A1, CYP1A2, and CYP1B1, as described by Chang
T K and Waxman D J in Method Mol. Biol., 320, 85-90, 2006. A number
of fluorgenic and luminogenic probe substrates are available to
assess cytochrome P450 activity with high sensitivity but they
exhibit broad specificity and as such are metabolised by a range of
cytochrome P450 enzymes in the CYP1, CYP2, and CYP3 families. For
example, Cali et al., Expert Opin. Drug Toxicol., 2(4): 62-45. 2006
describes the use of luminogenic substrates which couple to firefly
luciferase luminescence in a technology called P450-Glo. Another
example, is 7-ethoxycoumarin which undergoes cytochrome
P450-catalyzed 7-ethoxycoumarin O-deethylation to release the
highly fluourescent anion as described by Waxman D J and Change T K
H in "The use of 7-ethoxycoumarin to minitor multiple enzymes in
the human CYP1, CYP2, CYP3 families" in Methods in Molecular
Biology, vol. 320, Cytochrome P450 Protocols, Second Edition,
edited by Phillips I R and Shephard, E A, 2006.
[0016] Everett et al., Biochem. Pharmacol., 63, 1629-39, 2002
describe the reductive fragmentation of model indolequinone
prodrugs by cytochrome P450 reductase (not to be confused with
cytochrome P450s) in anoxia to release the
7-hydroxy-4-methylcoumarin anion. The model prodrug was
non-fluorescent at the pre-selected emission wavelength and
reductive fragmentation could be accurately measured by monitoring
the production of the coumarin anion (.lamda..sub.ex=380
nm/.lamda..sub.em=450 nm) using kinetic spectrofluorimetry.
[0017] Interactions between a limited number of compounds
(typically <100) and cytochrome P450s isozymes have been
described but results from such studies are difficult to compare
because of the differences in technologies, assay conditions and
data analysis methods as described by Rendic, S. "Summary of
information on human CYP enzymes: human P450 metabolism data" in
Drug Metab. Rev., 34, 83-448, 2002. Mnay computational strategies
have been advanced to generate predictive cytochrome P450 isozyme
substrate activity models but these are limited by a lack of a
single large, diverse data set of cytochrome P450 isozyme
activities as described by Veith et al., Nature Biotechnology, 27,
1050-55, 2009. The authors describe the construction of cytochrome
P450 bioactivity databases using quantitative high-throughput
screening (HTS) with a bioiluminescent enzyme substrate inhibition
assay to screen 17,143 chemical compounds against five cytochrome
P450 isozymes (CYP1A2, 2C9, 2C19, 2D6, and 3A4) expressed in normal
tissues mainly the liver and responsible for so-called phase 1
metabolism of drugs. It was concluded that the database should aid
in constructing and testing new predictive models for cytochrome
P450 activity to aid early stage drug discovery efforts.
[0018] Jensen et al., J. Med. Chem., 50, 501-11, 2007 describe the
methods for the in silico prediction of CYP2D6 and CYP3A4
inhibition based on a novel Gaussian Kernel weighted k-nearest
neighbour (k-NN) algorithm based on Tanimoto similarity searches on
extended connectivity fingerprints. The data set included modelling
of 1153 and 1182 drug candidates tested for CYP2D6 and CYP3A4
inhibition in human liver microsomes. For CYP2D6, 82% of the
classified test compounds were predicted to the correct class and
CYP3A4, 88% of the classified test compounds were correctly
classified.
[0019] Theoretically it may be possible to use cytochrome P450 HTS
to build a large database of bioactivities for tumour and normal
tissue cytochrome P450s and then develop a substrate prediction
model as a basis for the design and synthesis of selective
CYP1B1-activated prodrugs while screening out for pharmacological
liabilities associated with Phase 1 metabolism by normal tissue
cytochrome P450s. However, the reduction to practice is not obvious
from prior art and has to be rationalised against prodrug structure
and mechanism of conversion to the active drug when activated by
tumour-expressing cytochrome P450s.
[0020] Utilization of so-called `trigger-linker-effector` chemistry
in prodrug design requires the activation of the trigger to
initiate the fragmentation of a linker to release an effector
(typically an active drug), the biological activity of which is
masked in the prodrug form. The modular design of selective
prodrugs targeted at tumour-expressing cytochrome P450s such as
CYP1B1 require (1) the identification of selective trigger
moieties, (2) the use of bio-stable linkers which fragment
efficiently following trigger activation (usually by aromatic
hydroxylation), and (3) suitable effectors or drugs which do not
interfere with the efficiency of the triggering process.
[0021] CYP1B1 mRNA is expressed constitutively in all normal
extrahepatic human tissues, though the protein is usually
undetectable. In contrast, CYP1B1 protein is expressed at high
levels in tumours. It is understood that for a large range of
established or immortilized tumour cell lines (such as the MCF-7
breast cancer cells) originating from humans which have undergone
significant passaging in vitro but does not constitutively express
active CYP181 protein. Although CYP1B1 is not constitutively
expressed in MCF-7 breast tumour cells it is possible to induce
CYP1 enzyme expression both at the mRNA and protein level by
treating with aryl hydrocarbon agonists such as the dioxin
TCDD.
[0022] WO 99/40944 describes prodrugs that comprise a drug moiety
bound to a carrier framework, the prodrug being described activated
as though hydroxylation by CYP1B1 to release the drug moiety.
SUMMARY
[0023] We have surprisingly found that the compounds described
herein, distinct over those described in WO 99/40944, are broken
down in certain cells, in particular those that express cytochrome
P450 1B1 (hereinafter CYP1B1), but not in normal cells, as a
consequence of the compounds collapsing upon hydroxylation (e.g.
effected by CYP1B1-expressing cells), and in particular by
cancerous cells.
[0024] According to a first aspect therefore the present invention
provides a compound of formula (I):
##STR00001##
(wherein:
[0025] X.sup.1 is such that --X--X.sup.2 is --O--X.sup.2,
--S--X.sup.2, --SO.sub.2--O--X.sup.2, --SO.sub.2NZ.sup.10--X.sup.2,
conjugated alkenemethyloxy, conjugated alkenemethylthio, conjugated
alkenemethylSO.sub.2--O, conjugated alkenemethyl-SO.sub.2NZ.sup.10
or of the formula:
##STR00002##
[0026] --X.sup.2 is absent or is such that
X.sup.1--X.sup.2-Effector is one of
##STR00003##
[0027] each n and m is independently 0 or 1;
[0028] p is 0, 1 or 2;
[0029] X.sup.3 is oxygen or sulfur and additionally, when m=0, may
be SO.sub.2--O, SO.sub.2NZ.sup.10, conjugated alkenemethyloxy,
conjugated alkenemethylthio, conjugated alkenemethyl-SO.sub.2--O or
conjugated alkenemethyl-SO.sub.2NZ.sup.10;
[0030] each of Y.sup.1, Y.sup.2 and Y.sup.3 is independently carbon
or nitrogen, wherein if Y.sup.1 is nitrogen, Z.sup.1 is absent, if
Y.sup.2 is nitrogen, Z.sup.3 is absent and if Y.sup.3 is nitrogen,
Z.sup.5 is absent;
[0031] Y.sup.4 is an oxygen, carbon or nitrogen atom, sulfoxide or
sulfone;
[0032] --Y.sup.5-- is either (i) a single bond, (ii)=CH--, wherein
the double bond = in .dbd.CH-- is connected to Y.sup.4, or (iii)
--CH.sub.2-- or --CH.sub.2CH.sub.2--, or one of (ii) to (iii)
wherein the hydrogen atom in (ii) is or one or more hydrogen atoms
in (iii) are replaced with a substituent Z.sup.11, wherein Z.sup.11
is selected independently from alkyl, alkenyl, alkynyl, aryl,
aralkyl, alkyloxy, alkenyloxy, alkynyloxy, aryloxy, aralkyloxy,
alkylthioxy, alkenylthioxy, alkynylthioxy, arylthioxy,
aralkylthioxy, amino, hydroxy, thio, halo, carboxy, formyl, nitro
and cyano;
[0033] each of Z.sup.1--Z.sup.4, where present, are independently
selected from hydrogen, alkyl, alkenyl, alkynyl, aryl, aralkyl,
alkyloxy, alkenyloxy, alkynyloxy, aryloxy, aralkyloxy, alkylthioxy,
alkenylthioxy, alkynylthioxy, arylthioxy, aralkylthioxy, amino,
hydroxy, thio, halo, carboxy, formyl, nitro and cyano; and Z.sup.5,
where present, is independently selected from hydrogen alkyl,
alkenyl, alkynyl, aryl, aralkyl, alkyloxy, alkenyloxy, alkynyloxy,
aryloxy, aralkyloxy, alkylthioxy, alkenylthioxy, alkynylthioxy,
arylthioxy, aralkylthioxy, amino, hydroxy, thio, carboxy, formyl,
nitro and cyano, or one of Z.sup.2 & Z.sup.3, Z.sup.3 &
Z.sup.4 and Z.sup.4 and Z.sup.5 together with the atoms to which
they are connected form an aromatic ring fused to the remainder of
the compound, provided that at least one of Z.sup.1, Z.sup.2 and
Z.sup.4 is hydrogen;
[0034] Z.sup.6 is selected from hydrogen, alkyl, alkenyl, alkynyl,
aryl and aralkyl;
[0035] none, one or two of Y.sup.6 may be nitrogen atoms with the
remainder being carbon atoms;
[0036] each Z.sup.7 is independently hydrogen, alkyl or aryl;
[0037] each Z.sup.8 is independently selected from hydrogen, an
electron withdrawing group, unsubstituted C.sub.1-C.sub.6 alkyl,
substituted C.sub.1-C.sub.6 alkyl, unsubstituted C1-C.sub.6 alkoxy,
and substituted C.sub.1-C.sub.6 alkoxy where the substituted alkyl
or alkoxy are substituted with one or more groups selected from
ether, amino, mono- or di-substituted amino, cyclic C.sub.1-C.sub.5
alkylamino, imidazolyl, C.sub.1-6 alkylpiperazinyl, morpholino,
thiol, thioether, tetrazole, carboxylic acid, ester, amido, mono-
or di-substituted amido, N-connected amide, N-connected
sulfonamide, sulfoxy, sulfonate, sulfonyl, sulfoxy, sulfinate,
sufinyl, phosphonooxy, phosphate and sulfonamide;
[0038] each Z.sup.9 is independently oxygen or sulfur;
[0039] Z.sup.10 is hydrogen or alkyl, for example a C.sub.1-4
alkyl;
[0040] Effector is a molecule having a pharmacological, diagnostic
or screening function),
[0041] or a pharmaceutically acceptable salt, ester, amide or
solvate thereof.
[0042] Viewed from a second aspect, the invention provides a
composition comprised of a compound according to the first aspect
of the invention, or a pharmaceutically acceptable salt, ester,
amide or solvate thereof, together with a pharmaceutically
acceptable carrier.
[0043] Viewed from a third aspect the invention provides a compound
according to the first aspect of the invention, or a
pharmaceutically acceptable salt, ester, amide or solvate thereof,
for use as a medicament.
[0044] Viewed from a fourth aspect, the invention provides a
compound according to the first aspect of the invention, or a
pharmaceutically acceptable salt, ester, amide or solvate thereof,
for use in a method of treatment or prophylaxis of a proliferative
condition.
[0045] Viewed from a fifth aspect, the invention provides a method
of treatment or prophylaxis of a proliferative condition, said
method comprising administering a therapeutically or
prophylactically useful amount of a compound according to the first
aspect of the invention, or pharmaceutically acceptable salt,
ester, amide or solvate thereof, to a subject in need thereof.
[0046] Viewed from a sixth aspect, the invention provides the use
of a compound according to the first aspect of the invention or a
pharmaceutically acceptable salt, ester, amide or solvate thereof,
for the preparation of medicament for use in a method of treatment
or prophylaxis of a proliferative condition.
[0047] Viewed from a seventh aspect, the invention provides a
method of identifying a compound that is specifically activated by
a cytochrome P450 enzyme, said method comprising the steps of:
[0048] (a) contacting a set of compounds, according to the first
aspect of the invention in which Effector is a fluorophore, with
said cytochrome P450 enzyme and determining if said contact results
in release of said fluorophore from one or more compounds of said
set;
[0049] (b) contacting said set of compounds with a control tissue,
tissue or cell extract, or enzyme and determining if said contact
results in release of said fluorophore from one or more compounds
of said set; and
[0050] (c) identifying said compound specifically activated by said
cytochrome P450 as any compound in said set of compounds that
releases said fluorophore in step (a) but not, or only to a much
lesser extent, in step (b).
[0051] Viewed from an eighth aspect, the invention provides a
method for determining whether a compound of the invention, wherein
Effector is a molecule having a pharmacological function, is
efficacious in treating cancer, said method comprising
administering said compound to an animal having cancer, wherein
said cancer is resultant from implantation of either a recombinant
cell modified so as to express constitutively a cytochrome P450
enzyme, a tissue taken directly from a tumor or a cancer, or a cell
from an early passage cell line derived from a tissue taken
directly from a tumor or a cancer that expresses said cytochrome
P450 enzyme at levels similar to those from the tumor or cancer
from which it originates.
[0052] Further aspects and embodiment of the invention will follow
from the discussion that follows below.
BRIEF DESCRIPTION OF THE FIGURES
[0053] FIG. 1 depicts Western blots showing the detection of CYP1B1
expression in a transfected CHO/CYP1B1/CPR cell line (panel A) and
CYP1A1 expression in a transfected CHO/CYP1A1/CPR cell line (panel
B). Details are provided in the experimental section below.
[0054] FIG. 2a shows a mechanism for CYP1B1-induced 3-hydroxylation
of a compound of the invention (referred to herein as SU025-04)
followed by spontaneous release of a cytotoxic Effector molecule
(N,N'-bis(2-chloroethyl)phosphorodiamidate (also known as IPM
chloride) by 1,4 elimination.
[0055] FIG. 2b shows a mechanism for CYP1B1-induced 4-hydroxylation
of a compound of the invention (referred to herein as SU025-04)
followed by spontaneous release of a cytotoxic Effector molecule
(N,N'-bis(2-chloroethyl)phosphordiamidate (also known as IPM
chloride) by 1,6 elimination.
[0056] FIG. 2c shows a mechanism for CYP1B1-induced 6-hydroxylation
of a compound of the invention (referred to herein as SU025-04)
followed by spontaneous release of a cytotoxic Effector molecule
(N,N'-bis(2-chloroethyl)phosphordiamidate (also known as IPM
chloride) by 1,8 elimination.
[0057] FIG. 3 shows a mechanism for CYP1B1-induced 6-hydroxylation
of a compound of the invention (referred to herein as SU024-1-03)
followed by spontaneous release of an Effector molecule by 1,8
elimination.
DETAILED DESCRIPTION
[0058] The present invention arises from the provision of prodrugs
in which a so-called Effector molecule, which may be a cytostatic,
cytotoxic, diagnostic or screening molecule as described in greater
detail hereinafter, is chemically modified by reacting it whereby
to form a compound of formula (I). We have found that hydroxylation
of compounds of formula (I), in particular CYP1B1-induced
hydroxylation, allows release of the Effector molecules by a
collapse of the compounds of formula (I) which happens
spontaneously upon direct hydroxylation or hydroxylation via
epoxide formation.
[0059] In overview, the structure of the compounds of formula (I)
may be considered to comprise three parts: a trigger region, a
linker and an Effector molecule. The trigger serves as a substrate
for the typically CYP1B1-induced hydroxylation and may be generally
understood to comprise the bicyclic moiety depicted on the left
hand side of formula (I) and the substituents thereof, i.e.
comprising that part of the compounds containing Y.sup.1--Y.sup.5,
Z.sup.1--Z.sup.6 and the remaining carbon atoms to which some of
these moieties are attached. The trigger region of the compounds is
attached through a linking region comprising the
C(Z.sup.7)--X.sup.1--X.sup.2 unit to the Effector molecule which is
labelled as such.
[0060] The make-up and variability of these three regions--the
trigger, linker and Effector regions--of the compounds of formula
(I) are now described.
[0061] In the discussion that follows, reference is made to a
number of terms, which are to be understood to have the meaning
provided, below, unless the context dictates to the contrary.
[0062] By alkyl is meant herein a saturated hydrocarbyl radical,
which may be straight-chain, cyclic or branched (typically
straight-chain unless the context dictates to the contrary). Where
an alkyl group has one or more sites of unsaturation, these may be
constituted by carbon-carbon double bonds or carbon-carbon triple
bonds. Where an alkyl group comprises a carbon-carbon double bond
this provides an alkenyl group; the presence of a carbon-carbon
triple bond provides an alkynyl group. Typically alkyl, alkenyl and
alkynyl groups will comprise from 1 to 25 carbon atoms, more
usually 1 to 10 carbon atoms, more usually still 1 to 6 carbon
atoms it being of course understood that the lower limit in alkenyl
and alkynyl groups is 2 carbon atoms and in cycloalkyl groups 3
carbon atoms.
[0063] Alkyl, alkenyl or alkynyl groups may be substituted, for
example once, twice, or three times, e.g. once, i.e. formally
replacing one or more hydrogen atoms of the alkyl group. Examples
of such substituents are halo (e.g. fluoro, chloro, bromo and
iodo), aryl hydroxy, nitro, amino, alkoxy, alkylthio, carboxy,
cyano, thio, formyl, ester, acyl, thioacyl, amido, sulfonamido,
carbamate and the like.
[0064] By carboxy is meant herein the functional group CO.sub.2H,
which may be in deprotonated form (CO.sub.2.sup.-).
[0065] Halo is fluoro, bromo, chloro or iodo.
[0066] By acyl and thioacyl are meant the functional groups of
formulae --C(O)-alkyl or --C(S)-alkyl respectively, where alkyl is
as defined hereinbefore.
[0067] By ester is meant a functional group comprising the moiety
--OC(.dbd.O)--.
[0068] By amido is meant a functional group comprising the moiety
--N(H)C(.dbd.O)--; by carbamate is meant a functional group
comprising the moiety --N(H)C(.dbd.O)O--; and by sulfonamido is
meant a functional group comprising the moiety
--SO.sub.2N(H).sub.2--, in which each hydrogen atom depicted may be
replaced (independently in sulfonamido) with alkyl or aryl.
[0069] Alkyloxy (synonymous with alkoxy) and alkylthio moieties are
of the formulae --O-alkyl and --S-alkyl respectively, where alkyl
is as defined hereinbefore.
[0070] Likewise alkenyloxy, alkynyloxy, alkenylthio and alkynylthio
are of the formulae --O-alkenyl, -Oalkynyl, -Salkenyl and Salkynyl,
where alkenyl and alkynyl are as defined hereinbefore.
[0071] By amino group is meant herein a group of the formula
--N(R).sub.2 in which each R is independently hydrogen, alkyl or
aryl, e.g. an unsaturated, unsubstituted C.sub.1-6 alkyl such as
methyl or ethyl, or in which the two Rs attached to the nitrogen
atom N are connected. One example of this is whereby --R--R-- forms
an alkylene diradical, derived formally from an alkane from which
two hydrogen atoms have been abstracted, typically from terminal
carbon atoms, whereby to form a ring together with the nitrogen
atom of the amine. As is known the diradical in cyclic amines need
not necessarily be alkylene: morpholine (in which --R--R-- is
--(CH.sub.2).sub.2O(CH.sub.2).sub.2--) is one such example from
which a cyclic amino substituent may be prepared.
[0072] References to amino herein are also to be understood as
embracing within their ambit quaternised or protonated derivatives
of the amines resultant from compounds comprising such amino
groups. Examples of the latter may be understood to be salts such
as hydrochloride salts.
[0073] By aryl is meant herein a radical formed formally by
abstraction of a hydrogen atom from an aromatic compound.
[0074] Arylene diradicals are derived from aromatic moieties,
formally, by abstraction of two hydrogen atoms, and may be and
typically are, unless the context specifically dictates to the
contrary, monocyclic, for example, phenylene. As known to those
skilled in the art, heretoaromatic moieties are a subset of
aromatic moieties that comprise one or more heteroatoms, typically
O, N or S, in place of one or more carbon atoms and any hydrogen
atoms attached thereto. Exemplary heteroaromatic moieties, for
example, include pyridine, furan, pyrrole and pyrimidine. Further
examples of heteroaromatic rings include pyrdidyl, pyridazine (in
which 2 nitrogen atoms are adjacent in an aromatic 6-membered
ring); pyrazine (in which 2 nitrogens are 1,4-disposed in a
6-membered aromatic ring); pyrimidine (in which 2 nitrogen atoms
are 1,3-disposed in a 6-membered aromatic ring); or 1,3,5-triazine
(in which 3 nitrogen atoms are 1,3,5-disposed in a 6-membered
aromatic ring).
[0075] Aryl or arylene radicals may be substituted one or more
times with an electron-withdrawing group (for example a group
selected from halo, cyano (--CN), haloalkyl, amide, nitro, keto
(--COR), alkenyl, alkynyl, quarternary amino (--N.sup.+R.sub.3),
ester, amido (--CONR.sub.2), N-connected amido
(--NR--C(.dbd.O)--R), N-connected sulfonamido
(--NR--S(.dbd.O).sub.2R), sulfoxy (--S(.dbd.O).sub.2OH), sulfonate
(S(.dbd.O).sub.2OR), sulfonyl (S(.dbd.O).sub.2R) and sulfonamide
(--S(.dbd.O).sub.2--NR.sub.2), where (each) R is independently
selected from a C.sub.1-C.sub.6 alkyl group), a C.sub.3-C.sub.20
heterocyclic group, or a C.sub.3-C.sub.20 aryl group, typically a
C.sub.1-C.sub.6 alkyl group, unsubstituted C.sub.1-C.sub.6 alkoxy,
and substituted C.sub.1-C.sub.6 alkoxy where the substituted alkyl
or alkoxy are substituted with one or more groups selected from
ether, amino, mono- or di-substituted amino, cyclic C.sub.1-C.sub.5
alkylamino, imidazolyl, C.sub.1-C.sub.6 alkylpiperazinyl,
morpholino, thiol, thioether, tetrazole, carboxylic acid, ester,
amide, mono- or di-substituted amide, N-connected amide
(--NR--C(.dbd.O)--R), N-connected sulfonamide
(--NR--S(.dbd.O).sub.2--R), sulfoxy (--S(.dbd.O).sub.2OH),
sulfonate (S(.dbd.O).sub.2OR), sulfonyl (S(.dbd.O).sub.2R), sulfoxy
(S(.dbd.O)OH), sulfinate (S(.dbd.O)OR), sulfinyl (S(.dbd.O)R),
phosphonooxy(--OP(.dbd.O)(OH).sub.2), phosphate
(OP(.dbd.O)(OR).sub.2), and sulfonamide
(--S(.dbd.O).sub.2--NR.sub.2), where in (each) R is independently
selected from a C.sub.1-C.sub.6 alkyl group, a C.sub.3-C.sub.20
heterocyclic group, or a C.sub.3-C.sub.20 aryl group.
[0076] The trigger region of the compounds of formula (I) generally
comprises a bicyclic moiety comprising an aromatic ring (that
comprises the Y.sup.2 and Y.sup.3 moieties as indicated) fused to a
second ring (that comprises the Y.sup.1, Y.sup.4 and Y.sup.5
moieties that may be aromatic or non-aromatic.
[0077] Without being bound by theory, it is believed that the
activity of the compounds of formula (I) as substrates for
hydroxylation, e.g. effected by CYP1B1, is achieved in part by the
structure of the trigger moiety being susceptible to hydroxylation
when Z.sup.2 or Z.sup.4 is hydrogen, or when Y.sup.1--Z.sup.1 is
C--H, the hydroxylation thus taking place at one of the three
carbon atoms of those to which Z.sup.2 and Z.sup.4 are connected,
and Y.sup.1, where Y.sup.1 is carbon. As is depicted in FIG. 2,
hydroxylation at any of these positions in a representative
compound of the invention, labelled SU025-04, leads to spontaneous
collapse of the compound by an elimination process, either a 1,4-,
a 1,6- or a 1,8-elimination, depending upon at which of these
positions hydroxylation takes place.
[0078] It will be noted from the structure of the compounds of
formula (I) that, by virtue of the conjugation of carbon atoms to
which Z.sup.2 and Z.sup.4 are attached through Y.sup.1 to the
linker moiety, that any of the three mechanisms for spontaneous
breakdown of the compound may take place independently of the
nature of the Z.sup.6--Y.sup.4--Y.sup.5 region of the compounds.
Thus a wide variety to the nature of this region of the compounds
of formula (I) may be tolerated as discussed below. Also,
continuation of the region of conjugation is achieved inter alia by
the use of the conjugated X.sup.1 moieties described herein.
[0079] In the compounds of formula (I), each of the atoms indicated
by Y.sup.1, Y.sup.2 and Y.sup.3 may independently be a carbon atom
or a nitrogen atom. Where the atom concerned is a nitrogen atom,
the respective substituent (Z.sup.1, Z.sup.3 or Z.sup.5
respectively) will be absent. In certain embodiments of the
invention Y.sup.2 or Y.sup.3 is a carbon atom. In particular
embodiments of the invention both Y.sup.2 and Y.sup.3 are carbon
atoms. According to either of these embodiments--that in which both
Y.sup.2 or Y.sup.3 is a carbon atom or in which Y.sup.2 and Y.sup.3
are carbon atoms--or in which neither Y.sup.2 or Y.sup.3 is a
carbon atom, Y.sup.1 may be a carbon atom.
[0080] The substituents Z.sup.1, Z.sup.2 and Z.sup.4 may be
generally as described in claim 1. However, at least one of these
moieties is a hydrogen atom so as to allow a site for hydroxylation
of the compound. In some embodiments of the invention either
Z.sup.2 or Z.sup.4 is hydrogen. In other embodiments Z.sup.2 and
Z.sup.4 is hydrogen. In either of these embodiments--that in which
Z.sup.2 or Z.sup.4 is a hydrogen atom or in which both Z.sup.2 and
Z.sup.4 are hydrogen atoms--or in which neither Z.sup.2 or Z.sup.4
is a hydrogen atom, Z.sup.1 may be hydrogen. In certain embodiments
of the invention each of Z.sup.1, Z.sup.2 and Z.sup.4 is a hydrogen
atom.
[0081] Either Z.sup.3 or Z.sup.4 may, together with the adjacent
substituent on the aromatic ring (i.e. Z.sup.2 or Z.sup.4, or
Z.sup.3 or Z.sup.5 respectively) may, together with the atoms of
the aromatic ring to which these substituents are connected form an
aromatic ring fused to the remainder of the compound. Thus, Z.sup.2
and Z.sup.3, together with the carbon atom to which Z.sup.2 is
connected, and Y.sup.2, may form an aromatic ring. Similarly, for
example, Z.sup.4, Z.sup.5 and the carbon atom to which Z.sup.4 is
connected, and Y.sup.3, may together form an aromatic ring.
[0082] In certain embodiments of the invention, none or only two of
the pairs of substituents Z.sup.2 & Z.sup.3, Z.sup.3 &
Z.sup.4 and Z.sup.4 & Z.sup.5 together form a fused aromatic
ring. Thus, in certain embodiments there are no aromatic rings
fused to the aromatic ring comprising Y.sup.2 & Y.sup.3.
[0083] Specifically, substituents Z.sup.3 and Z.sup.5 are typically
not part of an aromatic ring fused to the remainder of the compound
of formula (I). Where this is the case, i.e. where these moieties
are individual substituents, Z.sup.3 may be alkyl, alkenyl,
alkynyl, aryl, aralkyl, alkyloxy, alkenyloxy, alkynyloxy, aryloxy,
aralkyloxy, alkylthioxy, alkenylthioxy, alkynylthioxy, arylthioxy,
aralkylthioxy, amino, hydroxy, thio, halo, carboxy, formyl, nitro
and cyano and Z.sup.5 may be alkyl, alkenyl, alkynyl, aryl,
aralkyl, alkyloxy, alkenyloxy, alkynyloxy, aryloxy, aralkyloxy,
alkylthioxy, alkenylthioxy, alkynylthioxy, arylthioxy,
aralkylthioxy, amino, hydroxy, thio, carboxy, formyl, nitro and
cyano. In certain embodiments of the invention, Z.sup.3 may be
alkyl, alkenyl, alkynyl, aryl, aralkyl, alkyloxy, alkenyloxy,
alkynyloxy, aryloxy, aralkyloxy, alkylthioxy, alkenylthioxy,
alkynylthioxy, arylthioxy, aralkylthioxy, amino, hydroxy, thio,
halo, carboxy, formyl, nitro and cyano.
[0084] In certain embodiments of the invention, Z.sup.3 and Z.sup.5
are individual substituents other than hydrogen atoms. Where
Z.sup.3 and Z.sup.5 are the same substituent or otherwise, Z.sup.3
and Z.sup.5 according to certain embodiments of the invention are
electron-donating groups such as alkoxy, alkylthioxy, aryloxy,
arylthioxy. In particular embodiments of the invention, Z.sup.3 or
Z.sup.5 are both amino or alkoxy, for example, C.sub.1-C.sub.6
alkoxy. Examples of such alkoxy groups include methoxy, ethoxy,
isopropoxy, n-propoxy and the like. In certain embodiments of the
invention either Z.sup.3 or Z.sup.5, or Z.sup.3 and Z.sup.5, are
methoxy. In certain embodiments of this invention Z.sup.3 and
Z.sup.5 are the same and are any of the immediately aforementioned
substituents, or classes of substituent. As noted above, the
compounds of formula (I) may be varied significantly in their
structure in the portion that comprises Z.sup.6--Y.sup.4--Y.sup.5.
Thus Y.sup.4 may be oxygen, sulfur, sulfoxide or sulfone whereupon
there is no Z.sup.6 substituent present (p=0), nitrogen (wherein
p=0 or 1) or a carbon atom whereupon p=1 or 2. In certain
embodiments of the invention p=0 and Y.sup.4 is oxygen, sulfur,
sulfone or sulfoxide. In particular embodiments of the invention
p=0 and Y.sup.4 is oxygen or sulfur. In certain embodiments of the
invention p=0 and Y.sup.4 is oxygen.
[0085] --Y.sup.5-- may be one of (i) a single bond, in which case
the trigger moiety is based upon the 6-membered aromatic Y.sup.2--
and Y.sup.3-containing ring fused to a 5-membered ring since in
this embodiment Y.sup.5 is effectively absent; or (ii)=CH-- in
which the double bond = is connected to Y.sup.4. In these
embodiments of the invention the trigger moiety is thus made up of
two fused aromatic rings and the skilled person will appreciate
that, where --Y.sup.5-- is .dbd.CH-- then Y.sup.4 is either a
nitrogen atom and p=0 or a carbon atom and p=1. Finally,
--Y.sup.5-- may be (iii) --CH.sub.2-- or --CH.sub.2CH.sub.2-- in
which case the trigger moiety comprises a bicyclic system
comprising a 6- or 7-membered ring fused to the aromatic 6-membered
ring substituted with Y.sup.2 and Y.sup.3. In certain embodiments
of the invention the or one or more of hydrogen or the hydrogen
atoms specified in options (ii) and (iii) for --Y.sup.5-- may be
replaced with a Z.sup.11 moiety, for example an alkyl or halo
moiety. In certain embodiments of the invention no Z.sup.11 is
present. In particular embodiments of the invention --Y.sup.5-- is
a single bond, for example wherein p=0 and Y.sup.4 is oxygen,
sulfur, sulfone or sulfoxide, p=0 and Y.sup.4 is oxygen or sulfur
and in particular wherein p=0 and Y.sup.4 is oxygen.
[0086] The linking moiety CH(Z.sup.7)--X.sup.1--X.sup.2 is now
described.
[0087] Z.sup.7 is hydrogen or an alkyl or aryl group, which, in
certain embodiments of the invention is unsubstituted. In certain
embodiments of the invention, the or each Z.sup.7 is an alkyl
group, e.g. an unsubstituted alkyl group such as an unsubstituted
C.sub.1-C.sub.6 alkyl group. Examples of Z.sup.7 moieties include
methyl and ethyl. In particular embodiments of the invention
Z.sup.7=hydrogen such that--CH(Z.sup.7)-- is methylene. In other
embodiments the or each Z.sup.7 moiety is a substituted alkyl
group, e.g. a substituted methyl or ethyl group. Examples of such
embodiments include amino-substituted alkyl groups, e.g. morpholino
or piperidinyl alkyl groups, or other groups that confer enhanced
water solubility. Alternatively the, each, or at least one Z.sup.7
may be an optionally substituted heteroaryl moiety such as
pyridyl.
[0088] X.sup.1 may be a variety of linking atoms or divalent
linking moieties, for example, X.sup.1 may be oxygen, sulfur,
sulfonamide or sulfonate ester. In addition, X.sup.1 may be
ethane-1,2-diylbis(methylcarbamate) or a conjugated alkenemethyloxy
moiety.
[0089] By a conjugated alkenemethyloxy moiety is meant a moiety of
the formula (.dbd.CH--CH).sub.q.dbd.CH--CH.sub.2--O-- wherein q is
an integer from 0 to 6, for example from 0 to 3, e.g. 0 or 1. The
skilled person will understand that the oxygen atom depicted in the
alkenemethyloxy moieties may be substituted with a sulfur atom
SO.sub.2--O or SO.sub.2NZ.sup.10 moiety, whereby to provide
conjugated alkenemethyl sulfonate or conjugated alkenemethyl
sulfonamide moieties as recited hereinbefore in which the oxygen or
sulfur atoms, or sulfonate of sulfonamide moieties (SO.sub.2--O and
SO.sub.2--NZ.sup.10) are attached to X.sup.2 or, if this is absent,
Effector.
[0090] According to certain embodiments of the invention X.sup.1 is
oxygen or sulfur. In many embodiments of the invention X.sup.1 is
oxygen.
[0091] X.sup.2 is an optional additional linking moiety, which is
either absent or interposed between X.sup.1 and the Effector
moiety.
[0092] X.sup.2 may be comprised of a variety of moieties as
described herein or may be absent. In certain embodiments of the
invention X.sup.2 is absent or X.sup.1--X.sup.2-Effector is one
of
##STR00004##
[0093] For example, X.sup.2 may comprise an
arylene-CH(Z.sup.7)X.sup.3 moiety (hereinafter
--ArCH(Z.sup.7)X.sup.3-- moiety) and/or an amide moiety. Where
present, the --Ar--CH(Z.sup.7)X.sup.3-- moiety may be flanked by
one or two amide or thioamide groups (C(Z.sup.9)NH). If flanked by
one amide or thioamide group, this may be disposed directly between
the X.sup.1 moiety and the aromatic ring of the
--Ar--CH(Z.sup.7)X.sup.3 (wherein n=1) moiety or interposed between
X.sup.3 and the Effector moiety (wherein m=1). Alternatively, an
amide or thioamide group may be present in both or neither of these
positions. In certain embodiments of the invention n=0 and m=1.
When X.sup.2 comprises a --Ar--CH(Z.sup.7)X.sup.3-- moiety, whether
or not this is flanked by one or two amide or thioamide moieties,
the X.sup.1 moiety that is attached to the aromatic ring either
directly or indirectly through an amide or thioamide moiety may be
attached at either of the two positions in the aromatic ring that
are ortho to the CH(Z.sup.7)X.sup.3 moiety of the
--Ar--CH(Z.sup.7)X.sup.3 system or at the para position.
Engineering these points of attachment in the aromatic rings of the
X.sup.2 moieties that comprise Ar--CH(Z.sup.7)X.sup.3-- moieties
permits 1,4-, 1,6- or 1,8-elimination of the Effector molecule. It
will be understood that the arylene group present in certain
embodiments of X.sup.2 may be heteroaromatic, that is to say one or
two or atoms Y.sup.6 may be nitrogen atoms with the remainder being
carbon atoms. An example of such a heteroarylene moiety is
pyridylene, in which one Y.sup.6 is a nitrogen atom. In many
embodiments of the invention each Y.sup.6 where present is a carbon
atom.
[0094] When an arylene group is present in the X.sup.2 moiety this
may be substituted as indicated at any of the four positions (not
connecting the arylene group to the Effector and trigger termini of
the compounds of formula (I) that is) by substituents Z.sup.8 which
may be selected independently as defined in claim 1.
[0095] Where X.sup.2 comprises one or more amide or thioamide
moieties--CH(Z.sup.9)NH this is typically, where present, (each)
Z.sup.9 is oxygen whereby to provide one or more amide moieties
although, where more than one Z.sup.9 is present, each Z.sup.9 may
be selected independently.
[0096] Finally, the Effector part of the compounds of formula (I)
is the moiety which provides the desired targeted effect in cells,
typically those in which CYP1B1 is expressed. The Effector
component may be any molecule having a pharmacological diagnostic
or screening function when released from the compound of formula
(I). By pharmacological or diagnostic function is meant that the
effector component, when released, has a discernable
pharmacological or diagnostic effect on the cells in which it is
released.
[0097] It will be understood by those skilled in the art that the
Effector component (Effector) in the compounds of formula (I) when
released may comprise an atom described herein as part of
X.sup.1--e.g. as oxygen or sulfur atom, or part of X.sup.2, e.g.
X.sup.3, e.g. an oxygen or sulfur atom. However, it is to be
understood that the distinctions between the trigger, linker and
Effector portions of the compounds of formula (I) are made simply
to assist in the description of the compounds of the invention; the
skilled person will be aware that the Effector portion in the
compounds of the invention constitutes the bulk of the Effector
molecule that is released upon hyroxylation-induced breakdown but
that one or some of the atoms in the Effector molecule that is
released may be provided by atoms described herein as being
X.sup.1, part of X.sup.1 or X.sup.2 and indeed elsewhere (e.g.
hydrogen atoms picked up from water molecules). Alternatively the
Effector molecule may be attached to the remainder of the compounds
of formula (I) through keto or fomyl groups for example.
[0098] The Effector molecule, where this has a pharmacological
effect, may be, for example, any chemical that has a cytostatic or
cytotoxic effect upon the cell that serves to effect its release is
expressed (e.g. CYP1B1-expressing cells). As is known, a cytotoxic
molecule is a molecule that is toxic to cells whereas a cytostatic
agent is one that suppresses the growth and/or replication of
cells.
[0099] In certain embodiments of the invention the Effector
molecule is a cytotoxic agent. Examples of cytotoxic agents that
may be used include but are not limited to alkylating agents,
antimitotic agents, antifolates, antimetabolites, DNA-damaging
agents and enzyme inhibitors (e.g tyrosine kinase inhibitors).
Specific examples of possible cytotoxic drug moieties include but
are not limited to bis(haloethyl)phosphoroamidates,
cyclophosphamides, gemcitabine, cytarabine, 5-fluorouracil,
6-mercaptopurine, camptothecin, topotecan, doxorubicin,
daunorubicin duocarmycin, etoposide, duetoposide, combretastatin
A-4, vinblastine, vincristine, AQ4N, hydroxyurea, maytansines,
enediyenes, epothilones, taxanes, bleomycins, calicheamicins,
colchicine, dacarbazine, dactinomycin, epirubicin, epirubicin
derivatives, fludarabine, hydroxyureapentatostatin, methotraxate,
mitomycin, mitoxantrone, carboplatin, cisplatin, taxels,
6-thioguanine, vinca alkaloids, platinum coordination complexes,
anthracenediones, substituted ureas, methyl hydrazine derivatives,
and nitrogen mustards.
[0100] In certain embodiments of the invention, the Effector
molecule is a phosphoramide mustard, that is to say a phosphoric
acid derivative in which one or two, typically two, of the hydroxyl
groups of phosphoric acid are exchanged for a nitrogen mustard, or
an oxygen- or sulfur-containing analogue thereof, and optionally
the P(.dbd.O) replaced with P(.dbd.S). A nitrogen mustard herein is
defined as a non-specifically alkylating amine, structurally
related to mustard gas (1,5-dichloro-3-thiapentane), in which the
sulfur atom is replaced with a nitrogen atom and, optionally, one
chlorethyl side chain is replaced by a hydrogen atom or alkyl
group, or one or both terminal chloro substituents are replaced by
a leaving group such as bromo, iodo or mesylate
(--OSO.sub.2CH.sub.3). Examples of phosphoramide mustards include
the compounds known as phosphoramide mustard (PM) and
isophosphoramide mustard (IPM):
##STR00005##
[0101] Thus, it will be noted that the compound PM is an example,
as well as the name of the class, of compounds known as
phosphoramide mustards since it may be regarded as a derivative of
phosphoric acid in which one of the hydroxyl groups has been
exchanged for a nitrogen mustard (the other hydroxyl group being
exchanged for an amino group (NH.sub.2)).
[0102] In those embodiments of the invention in which the Effector
molecule is a phosphoramide mustard, in which one or two, typically
two, of the hydroxyl groups of phosphoric acid derivative are
exchanged for an oxygen- or sulfur-containing analogue of a of
nitrogen mustard, by this is meant analogues of phosphoramide
mustards in which the nitrogen mustard is replaced with an analogue
in which one chloroethyl arm is absent and the nitrogen atom
exchanged for a sulfur or an oxygen atom.
[0103] In a particular embodiment of the present invention the
Effector molecule is connected to the remainder of the compound
through an oxygen or sulfur atom and -Effector is of formula
(II):
##STR00006##
(wherein: [0104] Z.sup.1 is oxygen or sulfur; [0105] each X.sup.4
is independently oxygen, sulfur or NZ.sup.13 wherein each-Z.sup.13
is independently --(CH.sub.2).sub.2--Z.sup.14, -alkyl or -hydrogen;
and [0106] each Z.sup.14 is independently chloro, bromo, iodo, or
mesylate).
[0107] In certain embodiments of the invention, Z.sup.12 is oxygen.
In these and other specific embodiments, each X.sup.4 is the same.
In these and other specific embodiments, each X.sup.4 is NZ.sup.13.
In these and other specific embodiments, each Z.sup.1 is hydrogen.
In these and other specific embodiments of the invention, each
Z.sup.14 is the same and/or is bromo or chloro. In particular
embodiments of the invention, each Z.sup.14 present (which may be
two, three or four Z.sup.14 moieties) is bromo.
[0108] Alternatively, the Effector molecule may be one that fulfils
a diagnostic function, for example allowing identification, or a
fuller understanding of the nature, of a tumor in which, for
example, CYP1B1 is expressed. An example of a class of Effector
molecules that are diagnostic molecules are fluorophoric molecules.
These may be useful in the diagnosis of cancerous cells. Examples
of fluorophoric compounds include coumarins, resorufins,
fluoresceins and rhodamines and it is in fact through a number of
experiments conducted on compounds of the invention comprising
coumarins as the Effector molecule that the viability of the
present invention has been demonstrated (see the examples section
below).
[0109] It will thus be appreciated that the compounds of formula
(I) in which Effector fulfils a diagnostic function may be of use
in methods of diagnosis and such methods constitute further aspects
of the present invention. Therefore, the invention provides a
compound of formula (I), or a pharmaceutically acceptable salt,
ester, amide or solvate thereof, for use in a method of diagnosis
of a proliferative condition, for example pre-malignant or
malignant cellular proliferation, a cancer, a leukaemia, psoriasis,
a bone disease, a fibroproliferative disorder or artherosclerosis,
for example a proliferative condition selected from bladder, brain,
breast, colon, head and neck, kidney, lung, liver, ovarian,
prostate and skin cancer, said method comprising administering an
amount of a compound, or pharmaceutically acceptable salt, ester,
amide or solvate of formula (I) to a subject having or suspected of
having such a proliferative and monitoring for the distribution of
released Effector molecules in the subject whereby to allow a
diagnosis to be made.
[0110] Alternatively, the Effector may be one that fulfils a
screening function, for example as part of a model prodrug library
collection, in order to identify trigger and linker combinations
that fragment when activated by CYP1B1 and allelic variants
thereof. An example of a class of Effector molecules are
fluorophoric molecules. Examples of fluorophoric compounds include
the well-known coumarins, resorufins, fluoresceins, and rhodamines.
It is in fact through a number of experiments conducted on
compounds of the invention comprising coumarins as the Effector
molecule that the viability of the present invention has been
demonstrated (see Example 1 in the section below). It will thus be
appreciated that the compounds of formula (I) in which the effector
fulfils a screening function may be of use in identifying trigger
and linker combinations for the design and synthesis of prodrugs
activated by CYP1B1 and such methods constitute further aspects of
the present invention.
[0111] It can be thus appreciated that compounds of formula (I) in
which an effector fulfils a screening function as part of a model
prodrug library collection can be used in combination with
cytochrome P450 substrate prediction models to guide the design and
synthesis of prodrugs with selectivity for the example CYP1B1, and
allelic variants thereof such as CYP1B1*3. For the purpose of
clarity, the combination of the model prodrug library with the
substrate prediction model links substrate specificity to prodrug
activation and fragmentation by CYP1B1, which is a fundamental
design principle. Futhermore, it can be thus appreciated that
compounds of formula (I) in which the effector fulfils a screening
function can be used in combination with cytochrome P450 substrate
prediction models to guide the design and synthesis of prodrugs
which are not activated by normal tissue cytochrome P450s
exemplified by CYP1A1, CYP1A2, CYP2C9, CYP2C19, CYP2D6, and CYP3A4.
An example of a substrate prediction model is the Gaussian Kernel
weighted k-NN algorithm based on Tanimoto similarity searches on,
but not limited to, descriptors such as extended connectivity
fingerprints. Cytochrome P450 substrate prediction models for
prodrug design can be built on bioactivity databases derived from
cytochrome P450 HTS from structurally diverse compound collections.
It is in fact through a number of experiments conducted on
compounds of the invention comprising coumarins as the effector
molecule used in combination with a CYP1B1 substrate prediction
model that the viability of the present invention has been
demonstrated (see the Examples 1 and 2).
[0112] Alternatively, the Effector may be one that fulfils a
screening function as part of a model prodrug library collection in
order to identify trigger and linker combinations that fragment
when activated by CYP1B1 and/or other cytochrome P450s and allelic
variants thereof over-expressed in cancer and other proliferative
conditions. An example of a class of Effector molecules are
fluorophoric molecules. Examples of fluorophoric compounds include
coumarins, resorufins, fluoresceins and rhodamines. Examples of
cytochrome P450s other than CYP1B1 which are over-expressed in
cancer include CYP2A/2B, CYP2F1, CYP2R1, CYP2S1, CYP2U1, CYP2W1,
CYP3A5, CYP3A7, CYP4Z.sup.1, CYP26A1, and CYP51.
[0113] It can be thus appreciated that compounds of formula (I) in
which an effector fulfils a screening function aspart of a model
prodrug library collection can be used in combination with
cytochrome P450 substrate prediction models to guide the design and
synthesis of prodrugs with selectivity for CYP1B1 and/or other
cytochrome P450s and allelic variants thereof over-expressed in
cancer and other proliferative conditions. An example of a class of
Effector molecules are fluorophoric molecules. Examples of
fluorophoric compounds include coumarins, resorufins, fluoresceins
and rhodamines. Examples of cytochrome P450s other than CYP1B1
which are over-expressed in cancer include CYP2A/2B, CYP2F1,
CYP2R1, CYP2S1, CYP2U1, CYP2W1, CYP3A5, CYP3A7, CYP4Z.sup.1,
CYP26A1, and CYP51. An example of a substrate prediction model is
the Gaussian Kernel weighted k-NN algorithm based on Tanimoto
similarity searches on, but not limited to, descriptors such as
extended connectivity fingerprints. Cytochrome P450 substrate
prediction models for prodrug design can be built on bioactivity
databases derived from cytochrome P450 HTS from structurally
diverse compound collections.
[0114] According to the aspects and embodiments of the present
invention whereby the Effector fulfils a screening function, for
example according to the seventh aspect of the invention, a set of
compounds will typically comprise a plurality of compounds, for
example comprising at least 10, for example at least 20 compounds.
In certain embodiments, the set may comprise up to 100, 1000,
10,000 or even 100,000 compounds. Such sets of compounds, i.e.
pluralities of compounds according to the first aspect of the
invention wherein the Effector is a fluorophore, as well as other
pluralities of compounds in which the Effector is not so limited
and/or the compounds may be pharmaceutically acceptable salts,
esters, amides or solvates, constitute a still further aspect of
the present invention.
[0115] According to embodiments of the seventh aspect of this
invention, where a compound releases the fluorophore in step (a)
but not, or only to a much lesser extent, in step (b), by this is
meant that the P450 enzyme typically releases at least 10-fold,
e.g. at least 20-fold, more of said fluorophore in step (a) as
compared to step (b).
[0116] Where screening, e.g. according to embodiments of the
seventh aspect of this invention yields a hit, e.g. and typically a
compound that releases the fluorophore in step (a) but not, or only
to a much lesser extent, in step (b), the method of the seventh
aspect of the invention optionally includes additional the steps
of:
[0117] (d) modeling compounds identical in structure to those
identified in step (c) except that the fluorophore is replaced with
a molecule having a pharmacologic function for binding to an active
site of said cytochrome P450 enzyme; and
[0118] (e) synthesizing compounds modeled in step (d) that are
predicted to be substrates for said cytochrome P450 enzyme.
[0119] Alternatively, these steps ((d) and (e)) may be practised
independently to the mandatory steps of the seventh aspect of this
invention (i.e. (a)-(c)) and so constitute a still further
embodiment of the present invention.
[0120] Typically the cytochrome P450 enzyme is selected from the
group consisting of CYP1B1, CYP2S1, CYP2W1, CYP4Z.sup.1 and allelic
variants thereof, for example CYP1B1 and allelic variants thereof,
e.g. CYP1B1.
[0121] An aspect of the present invention is the use of primary
human tumour cell lines of early passage number <20 in vitro
derived from resected cancer specimens. The primary head and neck
squamous cell carcinoma cell lines UT-SCCs described in Examples 4
and 5 below constitutively express CYP1B1 at the mRNA and protein
level and can be transplanted subcutaneously into immune-deficient
mice, (for example nude or servere combined immune deficient SCID
mice) with high engraftment rates to generate primary human tumour
xenografts where the constitive expression of cytochrome P450
protein expression matches that of the originating tumour in the
patient. These primary human tumour xenograft models, by
maintaining cytochrome P450 mRNA/protein expression similarly to
the originating patient tumour can therefore be used to assess the
efficacy of a compound of the invention, wherein the Effector
moiety is an agent having pharmacologic activity, in treating
cancer. Furthermore, in the clinical context these primary human
tumour xenograft models can be used to check if responses of a
compound of the invention, wherein the Effector moiety is an agent
having pharmacologic activity, are correlated with clinical
responses and outcomes, indicating usefulness for personalized
chemotherapy. The primary human tumour models can also be used to
compare the efficacy of a compound of claim 1, wherein the Effector
moiety is an agent having pharmacologic activity with standard
chemotherapeutic regimens and therefore to identify the most
effective regimens for compounds of claim 1 alone or in combination
with other chemotherapeutic agents.
[0122] Furthermore, as part of this invention it is possible to
derive primary human tumour xenografts by directly implanting
tumour tissue taken directly resected from patients and implanting
subcutaneously into, for example, nude, SCID and nonobese
diabetic/servere combined immune deficient (NOD/SCID) mice. It is
possible to generate first generation primary human tumour
xenografts for a range of different cancers which will retain the
histological and genetic characteristics of the originating tumor
and as such will constitutively express CYP1B1 mRNA/protein at a
level similar to the originating tumour. These primary human tumour
xenograft models, by maintaining CYP1B1 mRNA/protein expression
similarly to the originating patient tumour can therefore be used
to assess the efficacy of a compound of the invention, wherein the
Effector moiety is an agent having pharmacologic activity, in
treating cancer. Furthermore, in the clinical context these primary
human tumour xenograft models can be used to check if responses of
a compound of claim 1, wherein the Effector moiety is an agent
having pharmacologic activity are correlated with clinical
responses and outcomes, indicating usefulness for personalized
chemotherapy. The primary human tumour models can also be used to
compare the efficacy of a compound of the invention, wherein the
Effector moiety is an agent having pharmacologic activity, with
standard chemotherapeutic regimens and therefore to identify the
most effective regimens for compounds of the invention alone or in
combination with other chemotherapeutic agents.
[0123] Where according to the eighth aspect of this invention, the
cancer is resultant from implantation of a cell from an early
passage cell line derived from a tissue taken directly from a tumor
or a cancer that expresses said cytochrome P450 enzyme at levels
similar to those from the tumor or cancer from which it originates,
levels may be considered to be similar if they are within 10% to
those from the tumor or cancer from which it originates, for
example within 5%.
[0124] For use according to the present invention, the compounds or
a physiologically acceptable salt, solvate, ester or amide thereof
described herein may be presented as a pharmaceutical formulation,
comprising the compound or physiologically acceptable salt, ester,
amide or other physiologically functional derivative thereof,
together with one or more pharmaceutically acceptable carriers
therefor and optionally other therapeutic and/or prophylactic
ingredients. Any carrier(s) are acceptable in the sense of being
compatible with the other ingredients of the formulation and not
deleterious to the recipient thereof.
[0125] Examples of physiologically acceptable salts of the
compounds according to the invention include acid addition salts
formed with organic carboxylic acids such as acetic, lactic,
tartaric, maleic, citric, pyruvic, oxalic, fumaric, oxaloacetic,
isethionic, lactobionic and succinic acids; organic sulfonic acids
such as methanesulfonic, ethanesulfonic, benzenesulfonic and
p-toluenesulfonic acids and inorganic acids such as hydrochloric,
sulfuric, phosphoric and sulfamic acids.
[0126] The determination of physiologically acceptable esters or
amides, particularly esters is well within the skills of those
skilled in the art.
[0127] It may be convenient or desirable to prepare, purify, and/or
handle a corresponding solvate of the compounds described herein,
which may be used in the any one of the uses/methods described. The
term solvate is used herein to refer to a complex of solute, such
as a compound or salt of the compound, and a solvent. If the
solvent is water, the solvate may be termed a hydrate, for example
a mono-hydrate, di-hydrate, tri-hydrate etc, depending on the
number of water molecules present per molecule of substrate.
[0128] It will be appreciated that the compounds of the present
invention may exist in various stereoisomeric forms and the
compounds of the present invention as hereinbefore defined include
all stereoisomeric forms and mixtures thereof, including
enantiomers and racemic mixtures. The present invention includes
within its scope the use of any such stereoisomeric form or mixture
of stereoisomers, including the individual enantiomers of the
compounds of formulae (I) or (II) as well as wholly or partially
racemic mixtures of such enantiomers.
[0129] It will also be understood by those skilled in the art that
anticancer prodrugs, such as those described herein, can be
targeted towards particular tumours by attachment of a
tumour-targetting moiety such as tumour-targetting peptide, for
example small peptides identified through the development of
phage-displayed peptide libraries. Such peptides or other moieties
may assist in the targeting of conjugates that comprise them to a
particular cancer, particularly a solid tumour. Accordingly, the
provision of such conjugates, i.e. of a compound of the invention
conjugated to a tumour-targeting moiety, forms a further aspect of
this invention as do compositions, uses and methods described
herein that comprise or involve use of such conjugates.
[0130] The compounds of the present invention may be prepared using
reagents and techniques readily available in the art and/or
exemplary methods as described hereinafter. It has been found that
compounds of the present invention exhibit cytotoxicity in cells
expressing CYP1B1 enzyme, but are substantially non-toxic in normal
cells that do not express CYP1B1. Compounds of the invention may
also exhibit cytotoxicity in cells expressing CYP1A1 enzyme. In
practice, therefore, the compounds of the invention are non-toxic
pro-drugs that are converted (typically by CYP1B1) into cytotoxic
agents.
[0131] Suitably, the compounds of the invention have a cytotoxicity
IC.sub.50 value as defined below or less than 10 .mu.M,
advantageously less than 5 .mu.M, for example less than 1.0 .mu.M
or 0.5 .mu.M.
[0132] In some embodiments, the cytotoxicity of a compound of the
invention may be measured by incubating the compound at different
serial dilutions with cells engineered to express CYP1B1. Suitably,
said cells may be Chinese Hamster Ovary (CHO) cells, which may
contain recombinant CYP1B1 and cytochrome P-450 reductase (CPR).
High levels of functional enzyme when co-expressed with human P-450
reductase may be achieved using dihydrofolate reductase (DHFR) gene
amplification. Typically, the engineered cells may be incubated
with the compound and, after a suitable period of time (e.g., 96
hours), further incubated (e.g., for 1.5 hours) with a suitable
assay reagent to provide an indication of the number of living
cells in culture. A suitable assay reagent is MTS (see below) which
is bioreduced by cells into a formazan product that is soluble in
tissue culture medium. The absorbance of the formazan product can
be directly measured at 510 nm, and the quantitative formazan
product as measured by the amount of absorbance at 490 nm or 510 nm
is directly proportional to the number of living cells in culture.
Detailed methods for determining the IC.sub.50 value of a compound
according to the invention are described in Example 3 below.
[0133] By way of comparison, the IC.sub.50 values of the compounds
of the invention may also be measured in cells (e.g., Chinese
Hamster Ovary cells) that do not contain CYP1B1, for example wild
type CHO cells. The compounds of the invention may suitably have a
fold selectivity for CYP1B1 expressing cells of at least 200, where
the "fold selectivity" is defined as the quotient of the IC.sub.50
value of a given compound in non-CYP1 expressing cells and the
IC.sub.50 value of the same compound in CYP1B1 expressing
cells.
[0134] In some embodiments, the cytotoxicity of a compound of the
invention may be also measured by incubating the compound at
different serial dilutions with primary head and neck tumour cells
derived from patients with head and neck squamous cell carcinoma as
described in Example 4.
[0135] In some embodiments, the in vivo efficacy of a compound of
the invention may be measured by implanting primary head and neck
squamous cell carcinoma tumour cells which constitutively express
CYP1B1 subcutaneously into the flank of a nude mouse to generate
primary human tumour xenograft models and measuring the effect of
prodrug treatment on tumour growth as described in Example 5.
[0136] As such, the present invention also embraces the use of one
or more of the compounds of the invention, including the
aforementioned pharmaceutically acceptable esters, amides, salts,
solvates and prodrugs, for use in the treatment of the human or
animal body by therapy, particularly the treatment or prophylaxis
of proliferative conditions such, for example, as proliferative
disorders or diseases, in humans and non-human animals, including
proliferative conditions which are in certain embodiments of the
invention characterised by cells that express CYP1B1. More
particularly, the invention comprehends the use of one or more of
the compounds of the invention for the treatment of cancers
characterised in certain embodiments of the invention by CYP1B1
expression.
[0137] By "proliferative condition" herein is meant a disease or
disorder that is characterised by an unwanted or uncontrolled
cellular proliferation of excessive or abnormal cells which is
undesired, such as, neoplastic or hyperplastic growth, whether in
vitro or in vivo. Examples of proliferative conditions are
pre-malignant and malignant cellular proliferation, including
malignant neoplasms and tumours, cancers, leukemias, psoriasis,
bone diseases, fibroproliferative disorders (e.g., of connective
tissues) and atherosclerosis.
[0138] Said proliferative condition may be characterised in certain
embodiments of the invention by cells that express CYP1B1.
[0139] Said proliferative condition may be selected from bladder,
brain, breast, colon, head and neck, kidney, lung, liver, ovarian,
prostate and skin cancer. In some embodiments, said proliferative
condition may comprise a solid tumour.
[0140] By "treatment" herein is meant the treatment by therapy,
whether of a human or a non-human animal (e.g., in veterinary
applications), in which some desired therapeutic effect on the
proliferative condition is achieved; for example, the inhibition of
the progress of the disorder, including a reduction in the rate of
progress, a halt in the rate of progress, amelioration of the
disorder or cure of the condition. Treatment as a prophylactic
measure is also included. References herein to prevention or
prophylaxis herein do not indicate or require complete prevention
of a condition; its manifestation may instead be reduced or delayed
via prophylaxis or prevention according to the present invention.
By a "therapeutically-effective amount" herein is meant an amount
of the one or more compounds of the invention or a pharmaceutical
formulation comprising such one or more compounds, which is
effective for producing such a therapeutic effect, commensurate
with a reasonable benefit/risk ratio.
[0141] The compounds of the present invention may therefore be used
as anticancer agents. By the term "anticancer agent" herein is
meant a compound that treats a cancer (i.e., a compound that is
useful in the treatment of a cancer). The anti-cancer effect of the
compounds of the invention may arise through one or more
mechanisms, including the regulation of cell proliferation, the
inhibition of angiogenesis, the inhibition of metastasis, the
inhibition of invasion or the promotion of apoptosis.
[0142] It will be appreciated that appropriate dosages of the
compounds of the invention may vary from patient to patient.
Determining the optimal dosage will generally involve the balancing
of the level of therapeutic benefit against any risk or deleterious
side effects of the treatments of the present invention. The
selected dosage level will depend on a variety of factors including
the activity of the particular compound, the route of
administration, the time of administration, the rate of excretion
of the compound, the duration of the treatment, other drugs,
compounds or materials used in combination and the age, sex,
weight, condition, general health and prior medical history of the
patient. The amount of compound(s) and route of administration will
ultimately be at the discretion of the physician, although
generally the dosage will be to achieve local concentrations at the
site of action so as to achieve the desired effect.
[0143] Administration in vivo can be effected in one dose,
continuously or intermittently throughout the course of treatment.
Methods of determining the most effective means and dosage of
administration are well known to a person skilled in the art and
will vary with the formulation used for therapy, the purpose of the
therapy, the target cell being treated, and the subject being
treated. Single or multiple administrations can be carried out with
the dose level and pattern being selected by the treating
physician.
[0144] Pharmaceutical formulations include those suitable for oral,
topical (including dermal, buccal and sublingual), rectal or
parenteral (including subcutaneous, intradermal, intramuscular and
intravenous), nasal and pulmonary administration e.g., by
inhalation. The formulation may, where appropriate, be conveniently
presented in discrete dosage units and may be prepared by any of
the methods well known in the art of pharmacy. Methods typically
include the step of bringing into association an active compound
with liquid carriers or finely divided solid carriers or both and
then, if necessary, shaping the product into the desired
formulation.
[0145] Pharmaceutical formulations suitable for oral administration
wherein the carrier is a solid are most preferably presented as
unit dose formulations such as boluses, capsules or tablets each
containing a predetermined amount of active compound. A tablet may
be made by compression or moulding, optionally with one or more
accessory ingredients. Compressed tablets may be prepared by
compressing in a suitable machine an active compound in a
free-flowing form such as a powder or granules optionally mixed
with a binder, lubricant, inert diluent, lubricating agent,
surface-active agent or dispersing agent. Moulded tablets may be
made by moulding an active compound with an inert liquid diluent.
Tablets may be optionally coated and, if uncoated, may optionally
be scored. Capsules may be prepared by filling an active compound,
either alone or in admixture with one or more accessory
ingredients, into the capsule shells and then sealing them in the
usual manner. Cachets are analogous to capsules wherein an active
compound together with any accessory ingredient(s) is sealed in a
rice paper envelope. An active compound may also be formulated as
dispersible granules, which may for example be suspended in water
before administration, or sprinkled on food. The granules may be
packaged, e.g., in a sachet.
[0146] Formulations suitable for oral administration wherein the
carrier is a liquid may be presented as a solution or a suspension
in an aqueous or non-aqueous liquid, or as an oil-in-water liquid
emulsion.
[0147] Formulations for oral administration include controlled
release dosage forms, e.g., tablets wherein an active compound is
formulated in an appropriate release-controlling matrix, or is
coated with a suitable release-controlling film. Such formulations
may be particularly convenient for prophylactic use.
[0148] Pharmaceutical formulations suitable for rectal
administration wherein the carrier is a solid are most preferably
presented as unit dose suppositories. Suitable carriers include
cocoa butter and other materials commonly used in the art. The
suppositories may be conveniently formed by admixture of an active
compound with the softened or melted carrier(s) followed by
chilling and shaping in moulds.
[0149] Pharmaceutical formulations suitable for parenteral
administration include sterile solutions or suspensions of an
active compound in aqueous or oleaginous vehicles.
[0150] Injectable preparations may be adapted for bolus injection
or continuous infusion. Such preparations are conveniently
presented in unit dose or multi-dose containers, which are sealed
after introduction of the formulation until required for use.
Alternatively, an active compound may be in powder form that is
constituted with a suitable vehicle, such as sterile, pyrogen-free
water, before use.
[0151] An active compound may also be formulated as long-acting
depot preparations, which may be administered by intramuscular
injection or by implantation, e.g., subcutaneously or
intramuscularly. Depot preparations may include, for example,
suitable polymeric or hydrophobic materials, or ion-exchange
resins. Such long-acting formulations are particularly convenient
for prophylactic use.
[0152] Formulations suitable for pulmonary administration via the
buccal cavity are presented such that particles containing an
active compound and desirably having a diameter in the range of 0.5
to 7 microns are delivered in the bronchial tree of the
recipient.
[0153] As one possibility such formulations are in the form of
finely comminuted powders which may conveniently be presented
either in a pierceable capsule, suitably of, for example, gelatin,
for use in an inhalation device, or alternatively as a
self-propelling formulation comprising an active compound, a
suitable liquid or gaseous propellant and optionally other
ingredients such as a surfactant and/or a solid diluent. Suitable
liquid propellants include propane and the chlorofluorocarbons, and
suitable gaseous propellants include carbon dioxide.
Self-propelling formulations may also be employed wherein an active
compound is dispensed in the form of droplets of solution or
suspension.
[0154] Such self-propelling formulations are analogous to those
known in the art and may be prepared by established procedures.
Suitably they are presented in a container provided with either a
manually-operable or automatically functioning valve having the
desired spray characteristics; advantageously the valve is of a
metered type delivering a fixed volume, for example, 25 to 100
microlitres, upon each operation thereof.
[0155] As a further possibility an active compound may be in the
form of a solution or suspension for use in an atomizer or
nebuliser whereby an accelerated airstream or ultrasonic agitation
is employed to produce a fine droplet mist for inhalation.
[0156] Formulations suitable for nasal administration include
preparations generally similar to those described above for
pulmonary administration. When dispensed such formulations should
desirably have a particle diameter in the range 10 to 200 microns
to enable retention in the nasal cavity; this may be achieved by,
as appropriate, use of a powder of a suitable particle size or
choice of an appropriate valve. Other suitable formulations include
coarse powders having a particle diameter in the range 20 to 500
microns, for administration by rapid inhalation through the nasal
passage from a container held close up to the nose, and nasal drops
comprising 0.2 to 5% w/v of an active compound in aqueous or oily
solution or suspension.
[0157] It should be understood that in addition to the
aforementioned carrier ingredients the pharmaceutical formulations
described above may include, an appropriate one or more additional
carrier ingredients such as diluents, buffers, flavouring agents,
binders, surface active agents, thickeners, lubricants,
preservatives (including anti-oxidants) and the like, and
substances included for the purpose of rendering the formulation
isotonic with the blood of the intended recipient.
[0158] Pharmaceutically acceptable carriers are well known to those
skilled in the art and include, but are not limited to, 0.1 M and
preferably 0.05 M phosphate buffer or 0.8% saline. Additionally,
pharmaceutically acceptable carriers may be aqueous or non-aqueous
solutions, suspensions, and emulsions. Examples of non-aqueous
solvents are propylene glycol, polyethylene glycol, vegetable oils
such as olive oil, and injectable organic esters such as ethyl
oleate. Aqueous carriers include water, alcoholic/aqueous
solutions, emulsions or suspensions, including saline and buffered
media. Parenteral vehicles include sodium chloride solution,
Ringer's dextrose, dextrose and sodium chloride, lactated Ringer's
or fixed oils. Preservatives and other additives may also be
present, such as, for example, antimicrobials, antioxidants,
chelating agents, inert gases and the like.
[0159] Formulations suitable for topical formulation may be
provided for example as gels, creams or ointments.
[0160] Liquid or powder formulations may also be provided which can
be sprayed or sprinkled directly onto the site to be treated, e.g.
a wound or ulcer. Alternatively, a carrier such as a bandage,
gauze, mesh or the like can be sprayed or sprinkle with the
formulation and then applied to the site to be treated.
[0161] Therapeutic formulations for veterinary use may conveniently
be in either powder or liquid concentrate form. In accordance with
standard veterinary formulation practice, conventional
water-soluble excipients, such as lactose or sucrose, may be
incorporated in the powders to improve their physical properties.
Thus particularly suitable powders of this invention comprise 50 to
100% w/w and preferably 60 to 80% w/w of the active ingredient(s)
and 0 to 50% w/w and preferably 20 to 40% w/w of conventional
veterinary excipients. These powders may either be added to animal
feedstuffs, for example by way of an intermediate premix, or
diluted in animal drinking water.
[0162] Liquid concentrates of this invention suitably contain the
compound or a derivative or salt thereof and may optionally include
a veterinarily acceptable water-miscible solvent, for example
polyethylene glycol, propylene glycol, glycerol, glycerol formal or
such a solvent mixed with up to 30% v/v of ethanol. The liquid
concentrates may be administered to the drinking water of
animals.
[0163] In general, a suitable dose of the one or more compounds of
the invention may be in the range of about 1 .mu.g to about 5000
.mu.g/kg body weight of the subject per day, e.g., 1, 5, 10, 25,
50, 100, 250, 1000, 2500 or 5000 .mu.g/kg per day. Where the
compound(s) is a salt, solvate, prodrug or the like, the amount
administered may be calculated on the basis the parent compound and
so the actual weight to be used may be increased
proportionately.
[0164] In some embodiments, the one or more compounds of the
present invention may be used in combination therapies for the
treatment of proliferative conditions of the kind described above,
i.e., in conjunction with other therapeutic agents. Examples of
such other therapeutic agents include but are not limited to
topoisomerase inhibitors, alkylating agents, antimetabolites, DNA
binders and microtubule inhibitors (tubulin target agents), such as
cisplatin, cyclophosphamide, doxorubicin, etoposide, irinotecan,
fludarabine, 5FU, taxanes or mitomycin C. Other therapeutic agents
will be evident to those skilled in the art. For the case of active
compounds combined with other therapies the two or more treatments
may be given in individually varying dose schedules and via
different routes.
[0165] The combination of the agents listed above with a compound
of the present invention would be at the discretion of the
physician who would select dosages using his common general
knowledge and dosing regimens known to a skilled practitioner.
[0166] Where a compound of the invention is administered in
combination therapy with one, two, three, four or more, preferably
one or two, preferably one other therapeutic agents, the compounds
can be administered simultaneously or sequentially. When
administered sequentially, they can be administered at closely
spaced intervals (for example over a period of 5-10 minutes) or at
longer intervals (for example 1, 2, 3, 4 or more hours apart, or
even longer period apart where required), the precise dosage
regimen being commensurate with the properties of the therapeutic
agent(s).
[0167] The compounds of the invention may also be administered in
conjunction with non-chemotherapeutic treatments such as
radiotherapy, photodynamic therapy, gene therapy, surgery and
controlled diets.
[0168] The invention is now illustrated with reference to the
following non-limiting examples:
Preparation of Compounds
General
[0169] .sup.1H, .sup.13C and .sup.31P nuclear magnetic resonance
(NMR) spectra were recorded in the indicated solvent on either a
Bruker Avance DPX 500 MHz or Bruker Avance 300 MHz spectrometer.
Chemical shifts are expressed in ppm. Signal splitting patterns are
described as singlet (s), broad singlet (bs), doublet (d), triplet
(t), quartet (q), multiplet (m) or combination thereof. Low
resolution electrospray (ES) mass spectra were recorded on a Bruker
MicroTof mass spectrometer, run in a positive ion mode, using
either methanol/water (95:5) or water acetonitrile (1:1)+0.1%
formic acid as a mobile phase. High resolution electrospray
measurements were performed on a Bruker Microtof mass spectrometer.
LC-MS analysis were performed with an Agilent HPLC 1100 (Phenomenex
Gemini Column 5.mu. C18 110 .ANG. 50.times.3.0 mm, eluted with (0
to 20% MeOH/H.sub.2) and a diode array detector in series with a
Bruker Microtof mass spectrometer. Column chromatography was
performed with silica gel (230-400 mesh) or RediSep.RTM.4, 12, 40
or 80 g silica prepacked columns. All the starting materials are
commercially available and were used without further purification.
All reactions were carried out under dry and inert conditions
unless otherwise stated.
[Compounds indicated below with a parenthetical dagger (.dagger.)
are not examples of the invention but are included for a better
understanding of it.]
1. Phosphoroamidate Mustard Prodrugs
TABLE-US-00001 [0170] Code Structure SU025-04 ##STR00007## SU046-04
##STR00008##
Synthesis of the Phosphoroamidate Prodrugs SU025-04 and
SU046-04
[0171] 1. Synthesis of the trigger component of the prodrugs
##STR00009##
2-Bromo-3,5-dimethoxybenzaldehyde (1)
[0172] 3,5-dimethoxybenzaldehyde (12.6 g, 76 mmol) was dissolved in
acetic acid (350 mL). The resulting colourless solution was cooled
to 0.degree. C. A solution of bromine (3.9 mL) in ethanoic acid (50
mL) was added dropwise over 1 h. Once the addition was complete the
ice bath was removed and the resulting pale green solution was
stirred overnight at room temperature. Cold water was added to the
solution. The resulting white solid was collected by vacuum
filteration and rinsed with water. The solid was then redissolved
in EtOAc and adsorbed on silica gel. The product was purified by
flash chromatography, eluting with hexane/EtOAc (4:1) to give 1
(12.5 g, 66%) as a white solid. m/z=345.98 (M+H). .sup.1H NMR (500
MHz, CDCl.sub.3): .delta.: 10.43 (1H, s, CHO), 7.06 (1H, s, ArH),
6.73 (1H, s, ArH), 3.93 (3H, s, CH.sub.3O), 3.86 (3H, s,
CH.sub.3O). .sup.13C NMR (500 MHz, CDCl.sub.3): .delta.: 192.09
(CHO), 159.92 (C-5), 157.02 (C-3), 134.67 (C-1), 109.12 (C-2),
105.83, 103.37 (C-4 & C-6), 56.60 (OMe), 55.82 (OMe).
2-Hydroxy-3,5-dimethoxybenzaldehyde (2)
[0173] Morpholine (2.05 g, 24 mmol) and THF (40 mL) were placed in
a three-necked, round bottomed flask equipped with a stirring bar,
septum cap, dropping funnel, thermometer, and argon inlet. The
flask was cooled in a dry ice-acetone bath to -50.degree. C., and a
solution of n-BuLi in hexane (1.6M, 15 mL, 24 mmol) was added all
at once. After 10 min a solution of 1 (4.9 g, 20 mmol) in THF (30
mL) was added dropwise via a syringe over a period of 4 min, and
the mixture was cooled to .about.-75.degree. C. over 20 min. n-BuLi
in hexane (1.6M, 20 mL, 32 mmol) was then added dropwise over 45
min, keeping the temperature at -75.degree. C. After complete
addition of n-BuLi the solution was stirred for 35 min. A solution
of nitrophenol (6.90 g, 46 mmol) in 10 mL THF was added from the
dropping funnel, keeping the temperature at -75.degree. C. The
resulting dark mixture was stirred at -75.degree. C. for 4 h and
then allowed to warm to room temperature. It was acidified to pH 1
with 6N HCl and stirred for 15 min. After dilution with brine (100
mL), THE was removed in vacuo. The aqueous solution was extracted
with diethyl ether (4.times.40 mL). The combined organic layers
were extracted with 2 N NaOH (3.times.40 mL). The combined NaOH
extracts were washed with diethyl ether (3.times.20 mL) and then
acidified to pH 1 with concentrated HCl. The resulting mixture was
extracted with CH.sub.2Cl.sub.2 (3.times.20 mL), and the combined
organic extracts were washed with brine, dried (MgSO.sub.4) and
adsorbed on silica gel. The product was purified by flash
chromatography, eluting with EtOAc/hexane (1:2). Pure 2 was
obtained (2.0 g, 55%) as a yellow solid. m/z=183.06 (M+H). .sup.1H
NMR (500 MHz, CDCl.sub.3): .delta.: 10.71 (1H, s, OH), 9.91 (1H, s,
CHO), 6.77 (1H, d, J.sub.4, 6=2.8 Hz, H-6), 6.61 (1H, d, J.sub.4,
6=2.8 Hz, H-4), 3.92 (3H, s, OMe), 3.84 (3H, s, OMe). .sup.13C NMR
CDEPT135 (500 MHz, CDCl.sub.3): .delta.: 196.11 (CHO), 107.93
(C-6), 103.90 (C-4), 56.29 (OMe), 55.83 (OMe).
2-(2,2-Diethoxyethoxy)-3,5-dimethoxybenzaldehyde (3)
[0174] To a stirred suspension containing 2 (1.1 g, 6.0 mmol) and
K.sub.2CO.sub.3 (1.0 g, 7.2 mmol) in DMF (100 mL),
bromoacetaldehyde diethyl acetal (0.93 mL, 6.0 mmol) was added
dropwise. The mixture was refluxed for 4 h. After cooling, the
precipitate was filtered off and the solvent was evaporated in
vacuo. The crude residue was adsorbed on silica gel and purified by
flash chromatography, eluting with hexane/EtOAc (4:1) to give 3
(1.2 g, 67%) as a clear oil. .sup.1H NMR (500 MHz, CDCl.sub.3):
.delta.: 10.50 (1H, s, CHO), 6.88 (1H, d, J.sub.4, 6=2.9 Hz, H-6),
6.74 (1H, d, J.sub.4, 6=2.9 Hz, H-4), 4.83 (1H, t, J.sub.4, 6=5.3
Hz, CH), 4.14 (2H, d, J.sub.4, 6=5.3 Hz, CH.sub.2), 3.88 (3H, s,
OMe), 3.83 (3H, s, OMe), 3.77-3.71) (2H, m, CH.sub.2CH.sub.3),
3.63-3.58 (2H, m, CH.sub.2CH.sub.3), 1.24 (6H, t, J=7.1 Hz,
2.times.CH.sub.3).
5,7-dimethoxybenzofuran-2-carbaldehyde (4)
[0175] A stirred solution of 3 (1.2 g, 4.0 mmol) in acetic acid (35
mL) was refluxed for 16 h. After cooling, the solution was
evaporated to dryness. The crude product was adsorbed on silica gel
and purified by flash chromatography, eluting with hexane/EtOAc
(2:1) to give 4 (230 mg, 28%) as a white solid. .sup.1H NMR (500
MHz, CDCl.sub.3): .delta.: 9.89 (1H, s, CHO), 7.50 (1H, s, H-3),
6.69 (1H, d, J.sub.4, 6=2.2 Hz, H-6), 6.64 (1H, d, J.sub.4, 6=2.2
Hz, H-4), 4.01 (3H, s, OMe), 3.87 (3H, s, OMe). .sup.13C NMR CDEPT
135, (500 MHz, CDCl.sub.3): .delta.: 179.91 (CHO), 153.37 (C-2),
116.10 (C-3), 101.80 (C-6), 94.90 (C-4), 56.20 (OMe), 55.88
(OMe).
(5,7-dimethoxybenzofuran-2-yl)methanol (5)
[0176] Compound 4 (460 mg, 2.23 mmol) was dissolved in THF (5 mL)
and EtOH (1 mL). NaBH.sub.4 (102 mg, 2.68 mmol) was added
portionwise at 0.degree. C., with vigorous stirring. The suspension
was stirred at 0.degree. C. for 15 min and then at room temperature
for 1 h. Solvents were evaporated off in-vacuo. The crude residue
was taken up in EtOAc and washed with water, brine and dried
(MgSO.sub.4). The residue was adsorbed on silica gel and purified
by flash chromatography, eluting with hexane/EtOAc (1:1) to give 5
(388 mg, 82%) as an oil. m/z=209.08 (M+H). .sup.1H NMR (500 MHz,
CDCl.sub.3): .delta.: 6.62 (1H, s, H-3), 6.60 (1H, s, H-6), 6.46
(1H, s, H-4), 4.76 (2H, s, 2-CH.sub.2), 3.99 (3H, s, OMe), 3.85
(3H, s, OMe). .sup.13C NMR (500 MHz, CDCl.sub.3): .delta.: 157.23
(C-5), 156.72 (C-1), 145.36 (C-7), 139.50 (C-1a), 129.38 (C-4a),
104.62 (C-3), 96.96 (C-6), 94.58 (C-4), 57.98 (2-CH.sub.2), 55.95
(OMe), 55.83 (OMe).
2. Synthesis of the Effector Components of the Prodrugs
##STR00010##
[0177] N,N-bis (2-chloroethyl)phosphonamidic Acid (6)
[0178] To a suspension of 2-chloroethylamine hydrochloride (7.2 g,
62 mmol) in CH.sub.2Cl.sub.2 (110 mL) was added POCl.sub.3 (2.84
mL, 31 mmol) over 15 min at -78.degree. C. with vigorous stirring,
followed by the addition of a solution of TEA (17.5 mL, 124 mmol)
in CH.sub.2Cl.sub.2 (30 mL) over 4 h. The reaction mixture was
stirred at -78.degree. C. for 1 h and then allowed to warm to room
temperature and stirred for 2 h. The resulting solid was filtered
and washed with cold EtOAc. The solid was discarded. The filtrate
was concentrated under vacuum to about 5 mL and EtOAc was added (10
mL). The resulting suspension was filtered and washed with EtOAc
(2.times.10 mL). The solid was again discarded. The filtrate was
concentrated under vacuum to dryness. The residue was then
dissolved in THF (7 mL) followed by addition of an aqueous NaBr
solution (NaBr (5 g) in 100 mL water) at 0.degree. C. over 20 min.
The mixture was then warmed to room temperature and stirred for 15
h in a water bath. A white solid precipitated from the reaction
mixture. The mixture was then kept at -20.degree. C. in the freezer
for 2 h. The crystalline solid was filtered and washed with cold
water (2.times.50 mL, 0.degree. C.) and cold EtOAc (2.times.50 mL,
0.degree. C.). After drying at room temperature under vacuum
overnight, the product 6 was obtained (1.8 g, 26%) as a white
solid. .sup.1H NMR (500 MHz, DMSO-d.sub.6): .delta.: 5.29 (3H, br,
OH & NH), 3.55 (4H, t, J=7.0 Hz, 2.times.CH.sub.2), 3.01 (4H,
dt, J=12.2, 7.0 Hz, 2.times.CH.sub.2). .sup.31P NMR (500 MHz,
DMSO-d.sub.6) .delta.: 12.28 ppm.
N,N-bis (2-bromoethyl)phosphonamidic Acid (7)
[0179] Compound 7 was synthesized using a similar method as above.
It was obtained in 18% yield (1.64 g). .sup.1H NMR (500 MHz,
DMSO-d.sub.6): .delta.: 6.08 (3H, s, OH & NH), 3.46 (4H, t,
J=7.0 Hz, 2.times.CH.sub.2), 3.01 (4H, dt, J=12.2, 7.0 Hz,
2.times.CH.sub.2). .sup.31P NMR (500 MHz, DMSO-d.sub.6) .delta.:
12.23 ppm.
3. Coupling Reaction for the Synthesis of SU025-04 and SU046-04
##STR00011##
[0180]
5,7-Dimethoxybenzofuran-2-yl)methylN,N'-bis(2-chloroethyl)phosphord-
iamidate (8) SU025-04
[0181] To a suspension of 5 (300 mg, 1.44 mmol), 6 (479 mg, 2.16
mmol) and PPh.sub.3 (565 mg, 2.16 mmol) in THF (20 mL) was added
DIAD (0.426 mL, 2.16 mmol), dropwise at 0.degree. C. The resulting
suspension was warmed to room temperature and stirred for 2 h. The
solvent was removed, and the residue was purified by flash
chromatography (70% acetone in toluene) to give 8 (250 mg, 42%) as
an oil. m/z=823.08 (2M+H). .sup.1H NMR (500 MHz, DMSO-d.sub.6):
.delta. 7.27 (2H, s, 2.times.NH), 6.75 (1H, s, ArH-3), 6.62 (1H, d,
J.sub.4, 6=2.3 Hz, ArH-4), 6.49 (1H, d, J.sub.4, 6=2.3 Hz, ArH-6),
5.13 (2H, d, J=9.3 Hz, 2H), 3.99 (3H, s, OMe), 3.86 (3H, s, OMe),
3.29 (4H, m, 2.times.CH.sub.2), .sup.31P NMR (500 MHz,
DMSO-d.sub.6) .delta.:14.76 ppm. HRMS: Calcd for
C.sub.15H.sub.21N.sub.2O.sub.5PCl.sub.2Na, 433.0463; found
433.0471.
5,7-Dimethoxybenzofuran-2-yl)methylN,N'-bis(2-bromoethyl)phosphordiamidate
(9) SU046-04
[0182] Compound 9 (SU046-04) was synthesized using a similar method
as above. It was obtained in 25% yield (10 mg). m/z=500.96 (M+H),
1000.93 (2M+H). .sup.1H NMR (500 MHz, CDCl.sub.3): .delta. 6.76
(1H, s, ArH-3), 6.61 (1H, d, J.sub.4, 6=2.2 Hz, ArH-4), 6.48 (1H,
d, J.sub.4, 6=2.2 Hz, ArH-6), 5.13 (2H, d, J=9.4 Hz, 2H), 3.99 (3H,
s, OMe), 3.86 (3H, s, OMe), 3.49-3.45 (4H, m, 2.times.CH.sub.2),
3.40-3.33 (4H, m, 2.times.CH.sub.2) 3.23 (2H, bs, NH). .sup.13C NMR
(500 MHz, CDCl.sub.3): .delta.: 156.98, 152.91, 145.58, 139.97,
129.06, 128.24, 107.45, 97.70, 94.56, 59.73, 56.00, 42.90, 34.76,
30.98. HRMS: Calcd for C.sub.15H.sub.21N.sub.2O.sub.5PBr.sub.2Na,
520.9453; found 520.9454.
2. Ether and Thioether-Linked Model Prodrugs
TABLE-US-00002 [0183] TLE- M2- SU010A ##STR00012## VG015- 05
##STR00013## VG016- 05 (.dagger.) ##STR00014## VG017- 05
##STR00015## VG027- 05 ##STR00016## VG029- 05 ##STR00017## VG035-
04 ##STR00018## VG028- 05 ##STR00019## VG035- 05 ##STR00020## TLE-
M1- SU001A (.dagger.) ##STR00021## VG040- 03 ##STR00022## TLE- M1-
SU004A (.dagger.) ##STR00023## VG039- 03 ##STR00024## SU06- 02
(.dagger.) ##STR00025## SU010- 02 (.dagger.) ##STR00026## VG033- 03
##STR00027## VG015- 04 (.dagger.) ##STR00028## VG014- 04
##STR00029## VG015-02 (.dagger.) ##STR00030##
Synthesis of Ether and Thioether Linked Prodrugs
7-(benzofuran-2-ylmethoxy)-4-methyl-2H-chromen-2-one (10)
TLE-M2-SU010A
##STR00031##
[0184] 2-(bromomethyl)benzofuran (10)
[0185] Benzofuran-2-yl methanol (1.0 g, 6.7 mmol) was dissolved in
toluene (50 mL) and pyridine (653 .mu.L, 8.1 mmol) was added. The
solution was cooled to 0.degree. C. PBr.sub.3 (760 .mu.L, 8.1 mmol)
was added dropwise over 15 min. The reaction mixture was then
brought up to room temperature and stirred for 1 h. The mixture was
washed with K.sub.2CO 3 solution and extracted with EtOAc
(3.times.30 mL). The EtOAc layer was washed with brine and dried
(MgSO.sub.4). The solvent was evaporated off in-vacuo and product
was purified by flash chromatography, eluting with hexane:EtOAc
(4:1) to give 10 (780 mg, 55%) as an oil. .sup.1H NMR (500 MHz,
CDCl.sub.3): .delta.: 7.57 (1H, d, J=7.75 Hz, H-4), 7.52 (1H, d,
J=8.40, H-7), 7.35 (1H, t, J=8.90, H-4), 7.27 (1H, t, J=8.9 Hz,
H-5), 6.79 (1H, s, H-3), 4.64 (2H, s, 2-CH.sub.2). .sup.13C NMR
CDEPT 135, (500 MHz, CDCl.sub.3): .delta.: 155.34 (C-2), 152.65
(C-7a), 129.08 (C-3a), 125.20 (C-6), 123.16 (C-5), 121.34 (C--C-4),
111.46 (C-7), 106.30 (C-3), 23.61 (CH.sub.2--Br).
7-(benzofuran-2-ylmethoxy)-4-methyl-2H-chromen-2-one (11)
TLE-M2-SU010A
[0186] Sodium ethoxide (77 mg, 1.13 mmol) was added to DMF (10 mL)
at 0.degree. C., and the suspension was stirred for 10 min.
7-hydroxy-4-methylcoumarin (200 mg, 1.13 mmol) was slowly added and
resulting mixture was stirred at this temp for 0.5 h. To this
mixture 10 (200 mg, 0.94 mmol) was added portionwise. Resulting
reaction mixture was stirred at room temperature for 2 h. DMF was
evaporated off in-vacuo and the residue was taken up in EtOAc, and
washed with brine, water and 1M NaOH (2.times.30 mL). The organic
layer was dried (MgSO.sub.4) and product was purified by flash
chromatography, eluting with hexane: EtOAc (2:1) to give 11 (35 mg,
12%) as a white solid. m/z=307 (M+H). H.sup.1 NMR (500 MHz,
DMSO-d.sub.6): .delta.=7.75-7.60 (2H, m, ArH), 7.40-7.25 (2H, m,
ArH), 7.23 (1H, s, ArH), 7.12 (2H, d, CH), 6.25 (1H, s, CH), 5.41
(2H, s, CH.sub.2), 2.38 (3H, s, CH.sub.3).
7-((5-fluorobenzofuran-2-yl)methoxy)-4-methyl-2H-chromen-2-one (16)
VG015-05
##STR00032##
[0187] 2-(2,2-Diethoxyethoxy)-5-fluorobenzaldehyde (12)
[0188] To a stirred suspension containing
2-hydroxy-5-fluorobenzaldehyde (500 mg, 3.57 mmol) and
K.sub.2CO.sub.3 (524 mg, 13.79 mmol) in DMF (10 mL),
bromoacetaldehyde diethyl acetal (0.6 mL, 3.93 mmol) was added
dropwise. The mixture was refluxed for 4 h. After cooling, the
precipitate was filtered off and the solvent was evaporated in
vacuo. The crude residue was adsorbed on silica gel and purified by
flash chromatography, eluting with hexane/EtOAc (4:1) to give 12
(300 mg, 33%) as an oil. .sup.1H NMR (500 MHz, CDCl.sub.3):
.delta.: 10.30 (1H, s, CHO), 7.31 (1H, d, J=7.85 Hz), 7.10 (1H, t,
J=7.80 Hz), 6.87 (1H, d, J=8.86 Hz), 4.75 (1H, s, CH), 3.97 (2H, d,
J=2.35 Hz, CH.sub.2), 3.67-3.64 (2H, m, CH.sub.2CH.sub.3),
3.53-3.50 (2H, m, CH.sub.2CH.sub.3), 1.11 (6H, t, J=6.00 Hz,
2.times.CH.sub.3).
5-fluorobenzofuran-2-carbaldehyde (13)
[0189] A stirred solution of 12 (300 mg, 4.0 mmol) in acetic acid
(10 mL) was refluxed for 24 h. After cooling, the solution was
evaporated to dryness. The crude product was adsorbed on silica gel
and purified by flash chromatography, eluting with hexane/EtOAc
(4:1) to give the 13 (180 mg, 94%) as a white solid, .sup.1H NMR
(500 MHz, CDCl.sub.3): .delta.: 9.89 (1H, s, CHO), 7.57 (2H, m,
ArH), 7.41 (1H, d, J=7.20 Hz), 7.26 (1H, d, J=6.94 Hz, H-4).
.sup.13C NMR CDEPT 135, (500 MHz, CDCl.sub.3): .delta.: 179.74
(CHO), 117.73 (C-3), 117.25 (C-7), 113.81 (C-6), 108.68 (C-4).
(5-fluorobenzofuran-2-yl)methanol (14)
[0190] Compound 13 (180 mg, 1.10 mmol) was dissolved in EtOH (12
mL). NaBH.sub.4 (45 mg, 1.21 mmol) was added portionwise at
0.degree. C., with vigorous stirring. The suspension was stirred at
0.degree. C. for 15 min and then at room temperature for 1.5 h.
Solvents were evaporated off in-vacuo. The crude residue was taken
up in EtOAc and washed with water, brine and dried (MgSO.sub.4).
The solvent was evaporated off in vacuo. The residue was adsorbed
on silica gel and purified by flash chromatography, eluting with
hexane/EtOAc (3:1) to give 14 (150 mg, 91%) as a white solid.
.sup.1H NMR (500 MHz, CDCl.sub.3): .delta.: 7.36 (1H, d, J=8.35 Hz,
H-7), 7.19 (1H, d, J=7.80, H-4), 7.00 (1H, t, J=8.75H-6), 6.60 (1H,
s, H-3), 4.75 (2H, s, CH.sub.2).
2-(bromomethyl)-5-fluorobenzofuran (15)
[0191] Compound 14 (150 mg, 0.90 mmol) was dissolved in toluene (10
mL) and the solution was cooled to 0.degree. C. PBr.sub.3 (102
.mu.L, 1.08 mmol) was added dropwise over 15 min. The reaction
mixture was then brought up to room temperature and stirred for 1
h. The solvent was evaporated off in-vacuo. The residue was
adsorbed on silica gel and purified by flash chromatography,
eluting with hexane/EtOAc (4:1) to give 15 (150 mg, 72%) as an oil.
.sup.1H NMR (500 MHz, CDCl.sub.3): .delta.: 7.43 (1H, d, J=7.60 Hz,
H-7), 7.21 (1H, t, J=8.10, H-4), 7.06 (1H, t, J=8.90, H-4), 6.75
(1H, s, H-3), 4.60 (2H, s, 2-CH.sub.2). .sup.13C NMR CDEPT 135,
(500 MHz, CDCl.sub.3): .delta.: 113.08 (C-7), 112.08 (C-6), 106.87
(C-4), 106.34 (C-3), 60.42 (CH.sub.2--Br).
7-((5-fluorobenzofuran-2-yl)methoxy)-4-methyl-2H-chromen-2-one (16)
VG015-05
[0192] Sodium ethoxide (8.9 mg, 0.13 mmol) was added to DMF (3 ml)
at 0.degree. C., and the suspension was stirred for 10 min.
7-hydroxy-4-methylcoumarin (25.4 mg, 0.14 mmol) was slowly added
and resulting mixture was stirred at this temp for 0.5 h. To this
mixture 15 (30 mg, 0.13 mmol) was added portionwise. Resulting
reaction mixture was stirred at room temperature for 2 h. DMF was
evaporated off in-vacuo and the residue was purified by flash
chromatography, eluting with hexane: EtOAc (3:1) to give 16 (9.0
mg, 21%) as a white solid. m/z=325.20 (M+H).
7-((5,7-difluorobenzofuran-2-yl)methoxy)-4-methyl-2H-chromen-2-one
(19) VG016-05
##STR00033##
[0193] 2-(2,2-diethoxyethoxy)-3,5-difluorobenzaldehyde (17)
[0194] To a stirred suspension containing
2-hydroxy-3,5-fluorobenzaldehyde (1.0 g, 6.32 mmol) and
K.sub.2CO.sub.3 (960 mg, 6.95 mmol) in DMF (10 mL),
bromoacetaldehyde diethyl acetal (1.07 mL, 6.95 mmol) was added
dropwise. The mixture was refluxed for 4 h. After cooling, the
precipitate was filtered off and the solvent was evaporated in
vacuo. The crude residue was adsorbed on silica gel and purified by
flash chromatography, eluting with hexane/EtOAc (4:1) to give 17
(380 mg, 22%) as an oil, .sup.1H NMR (500 MHz, CDCl.sub.3):
.delta.: 10.41 (1H, s, CHO), 7.29 (1H, d, J=6.80 Hz), 7.10 (1H, t,
J=8.30 Hz), 4.80 (1H, s, CH), 4.20 (2H, d, J=3.25 Hz, CH.sub.2),
3.71 (2H, t, J=7.10 Hz, CH.sub.2CH.sub.3), 3.57 (2H, t, J=7.45 Hz,
CH.sub.2CH.sub.3), 1.19 (6H, t, J=6.25 Hz, 2.times.CH.sub.3).
2-(bromomethyl)-5,7-difluorobenzofuran (18)
[0195] A stirred solution of 17 (380 mg, 1.39 mmol) in acetic acid
(10 mL) was refluxed for 24 h. After cooling, the solution was
evaporated to dryness. The crude product (300 mg) was dissolved in
EtOH (5 mL). NaBH.sub.4 (73 mg, 1.98 mmol) was added portionwise at
0.degree. C., with vigorous stirring. The suspension was stirred at
0.degree. C. for 15 min and then at room temperature for 1.5 h.
Solvents were evaporated off in-vacuo. The crude residue (280 mg)
was dissolved in toluene (20 mL) and the solution was cooled to
0.degree. C. PBr.sub.3 (142 .mu.L, 1.52 mmol) was added dropwise
over 15 min. The reaction mixture was then brought up to room
temperature and stirred for 1 h. The solvent was evaporated off
in-vacuo. The residue was adsorbed on silica gel and purified by
flash chromatography, eluting with hexane/EtOAc (4:1) to give 18
(210 mg, 61%). .sup.1H NMR (500 MHz, CDCl.sub.3): .delta.: 7.03
(1H, d, J=7.50 Hz, H-4), 6.87 (1H, t, J=9.80, H-5), 6.79 (1H, s,
H-3), 4.59 (2H, s, 2-CH.sub.2).
7-((5,7-difluorobenzofuran-2-yl)methoxy)-4-methyl-2H-chromen-2-one
(19) VG016-05
[0196] Sodium ethoxide (8.9 mg, 0.13 mmol) was added to DMF (3 mL)
at 0.degree. C., and the suspension was stirred for 10 min.
7-hydroxy-4-methylcoumarin (25.4 mg, 0.14 mmol) was slowly added
and resulting mixture was stirred at this temp for 0.5 h. To this
mixture 2-(bromomethyl)-5-fluorobenzofuran (30 mg, 0.12 mmol) was
added portionwise. Resulting reaction mixture was stirred at room
temperature for 2 h. DMF was evaporated off in-vacuo and the
residue was purified by flash chromatography, eluting with hexane:
EtOAc (3:1) to give 19 (8.8 mg, 21%) as a white solid. m/z=343.12
(M+H).
7-((5,7-difluorobenzofuran-2-yl)methoxy)-4-methyl-2H-chromen-2-one
(22)(VG017-05)
##STR00034##
[0197] 2-(2,2-diethoxyethoxy)-5-fluoro-3-methylbenzaldehyde
(20)
[0198] To a stirred suspension containing
5-fluoro-2-hydroxy-3-methylbenzaldehyde (1.0 g, 6.49 mmol) and
K.sub.2CO.sub.3 (980 mg, 7.10 mmol) in DMF (8 mL),
bromoacetaldehyde diethyl acetal (1.10 mL, 7.15 mmol) was added
dropwise. The mixture was refluxed for 4 h. After cooling, the
precipitate was filtered off and the solvent was evaporated in
vacuo. The crude residue was adsorbed on silica gel and purified by
flash chromatography. The product was eluted with hexane/EtOAc
(4:1) to give 20 (350 mg, 20%) as an oil, .sup.1H NMR (500 MHz,
CDCl.sub.3): .delta.: 10.40 (1H, s, CHO), 7.32 (1H, d, J=7.30 Hz,
ArH), 7.14 (1H, d, J=7.75 Hz, ArH), 4.85 (1H, s, CH), 3.95 (2H, s,
CH.sub.2), 3.75 (2H, t, J=7.15 Hz, CH.sub.2CH.sub.3), 3.61 (2H, t,
J=7.20 Hz, CH.sub.2CH.sub.3), 2.36 (3H, s, CH.sub.3), 1.24 (6H, t,
J=5.65 Hz, 2.times.CH.sub.3).
2-(bromomethyl)-5-fluoro-7-methylbenzofuran (21)
[0199] A stirred solution of 20 (350 mg, 1.30 mmol) in acetic acid
(10 mL) was refluxed for 24 h. After cooling, the solution was
evaporated to dryness. The crude product (300 mg) was dissolved in
THF (5 mL). NaBH.sub.4 (78 mg, 2.02 mmol) was added portionwise at
0.degree. C., with vigorous stirring. The suspension was stirred at
0.degree. C. for 15 min and then at room temperature for 1.5 h.
Solvents were evaporated off in-vacuo. The crude residue (260 mg)
was dissolved in toluene (20 mL) and the solution was cooled to
0.degree. C. PBr.sub.3 (135 .mu.L, 1.44 mmol) was added dropwise
over 15 min. The reaction mixture was then brought up to room
temperature and stirred for 1 h. The solvent was evaporated off
in-vacuo. The residue was adsorbed on silica gel and purified by
flash chromatography, eluting with hexane/EtOAc (4:1) to give 21
(200 mg, 36%). .sup.1H NMR (500 MHz, CDCl.sub.3): .delta.: 7.03
(1H, d, J=7.50 Hz, H-4), 6.88 (1H, t, J=9.80, H-5), 6.75 (1H, s,
H-3), 4.69 (2H, s, 2-CH.sub.2), 2.54 (3H, s, CH.sub.3).
7-((5,7-difluorobenzofuran-2-yl)methoxy)-4-methyl-2H-chromen-2-one
(22) VG017-05
[0200] Sodium ethoxide (8.9 mg, 0.13 mmol) was added to DMF (3 mL)
at 0.degree. C., and the suspension was stirred for 10 min.
7-hydroxy-4-methylcoumarin (25.4 mg, 0.14 mmol) was slowly added
and resulting mixture was stirred at this temp for 0.5 h. To this
mixture 21 (30 mg, 0.12 mmol) was added portionwise. Resulting
reaction mixture was stirred at room temperature for 2 h. DMF was
evaporated off in-vacuo and the residue was purified by flash
chromatography, eluting with hexane: EtOAc (3:1) to give 22 as a
white solid (11 mg, 26%). m/z=33.20 (M+H).
7-((5-methoxybenzofuran-2-yl)methoxy)-4-methyl-2H-chromen-2-one
(27) VG027-05
##STR00035##
[0201] 2-(2,2-diethoxyethoxy)-5-methoxybenzaldehyde (23)
[0202] To a stirred suspension containing
2-hydroxy-5-methoxybenzaldehyde (2.0 g, 13.16 mmol) and
K.sub.2CO.sub.3 (2.18 g, 15.79 mmol) in DMF (20 mL),
bromoacetaldehyde diethyl acetal (2.43 mL, 15.79 mmol) was added
dropwise. The mixture was refluxed for 4 h. After cooling, the
precipitate was filtered off and the solvent was evaporated in
vacuo. The crude residue was adsorbed on silica gel and purified by
flash chromatography. The product was eluted with hexane/EtOAc
(4:1) to give the target compound 23 (1.10 g, 31%) as an oil.
.sup.1H NMR (500 MHz, CDCl.sub.3): .delta.: 10.49 (1H, s, CHO),
7.33 (1H, d, J=3.30 Hz, ArH), 7.12 (1H, dd, J=5.75 & 3.30 Hz,
ArH), 6.97 (1H, d, J=9.05 Hz), 4.87 (1H, t, J=5.25 Hz, CH), 4.09
(2H, d, J=5.25 Hz, CH.sub.2), 3.81-3.78 (2H, m, CH.sub.2CH.sub.3),
3.67-3.64 (2H, m, CH.sub.2CH.sub.3), 1.26 (6H, t, J=7.05 Hz,
2.times.CH.sub.3).
5-methoxybenzofuran-2-carbaldehyde (24)
[0203] A stirred solution of 23 (1.0 g, 3.74 mmol) in acetic acid
(10 mL) was refluxed for 16 h. After cooling, the solution was
evaporated to dryness. The crude product was adsorbed on silica gel
and purified by flash chromatography, eluting with hexane/EtOAc
(4:1) to give 24 (160 mg, 24%) as a white solid. .sup.1H NMR (500
MHz, CDCl.sub.3): .delta.: 9.80 (1H, s, CHO), 7.49-7.45 (2H, m,
ArH), 7.12-7.09 (2H, m, ArH), 3.85 (3H, s, OCH).
(5-methoxybenzofuran-2-yl)methanol (25)
[0204] Compound 24 (3.5 g, 19.9 mmol) was dissolved in EtOH (20
mL). NaBH.sub.4 (957 mg, 25.87 mmol) was added portionwise at
0.degree. C., with vigorous stirring. The suspension was stirred at
0.degree. C. for 15 min and then at room temperature for 1.5 h.
Solvent was evaporated off in-vacuo. The residue was adsorbed on
silica gel and purified by flash chromatography, eluting with
hexane/EtOAc (2:1) to give 25 (3.0 g, 85%) as a white solid.
.sup.1H NMR (500 MHz, CDCl.sub.3): .delta.: 7.36 (1H, d, J=8.90 Hz,
H-7), 7.02 (1H, d, J=2.6, H-4), 6.90 (1H, dd, J=6.30 & 2.60,
H-6), 6.61 (1H, s, H-3), 4.76 (2H, s, 2-CH.sub.2), 3.86 (3H, s,
OCH.sub.3), 2.16 (1H, bs, OH). .sup.13C NMR CDEPT 135, (500 MHz,
CDCl.sub.3): .delta.: 113.07 (C-7), 111.69 (C-6), 104.34 (C-4),
103.60 (C-3), 58.24 (CH.sub.2), 55.92 (OCH.sub.3).
2-(bromomethyl)-5-methoxybenzofuran (26)
[0205] Compound 25 (40 mg, 0.22 mmol) was dissolved in toluene (5
mL) and the solution was cooled to 0.degree. C. PBr.sub.3 (21
.mu.L, 0.22 mmol) was added dropwise over 10 min. The reaction
mixture was then brought up to room temperature and stirred for 1
h. The solvent was evaporated off in-vacuo. The residue was
adsorbed on silica gel and purified by flash chromatography,
eluting with hexane/EtOAc (4:1) to give 26 (40 mg, 74%) as an oil.
.sup.1H NMR (500 MHz, CDCl.sub.3): .delta.: 7.39 (1H, d, J=8.90 Hz,
H-7), 7.01 (1H, d, J=2.55, H-4), 6.94 (1H, dd, J=6.35 & 2.60,
H-6), 6.72 (1H, s, H-3), 4.61 (2H, s, 2-CH.sub.2), 3.86 (3H, s,
OCH.sub.3).
7-((5-methoxybenzofuran-2-yl)methoxy)-4-methyl-2H-chromen-2-one
(27) VG027-05
[0206] Sodium ethoxide (12 mg, 0.18 mmol) was added to DMF (5 mL)
at 0.degree. C., and the suspension was stirred for 10 min.
7-hydroxy-4-methylcoumarin (32 mg, 0.17 mmol) was slowly added and
resulting mixture was stirred at this temp for 0.5 h. To this
mixture 26 (40 mg, 0.17 mmol) was added portionwise. Resulting
reaction mixture was stirred at room temperature for 2 h. DMF was
evaporated off in-vacuo and the residue was purified by flash
chromatography, eluting with hexane: EtOAc (3:1) to give 27 (17 mg,
30%) as a white solid. m/z=337.04 (M+H), 673.13 (2M+H).
7-((7-methoxybenzofuran-2-yl)methoxy)-4-methyl-2H-chromen-2-one
(31) VG029-05
##STR00036##
[0207] 2-(2,2-diethoxyethoxy)-3-methoxybenzaldehyde (28)
[0208] To a stirred suspension containing
2-hydroxy-3-methoxybenzaldehyde (4.0 g, 26.3 mmol) and
K.sub.2CO.sub.3 (4.36 g, 31.60 mmol) in DMF (15 mL),
bromoacetaldehyde diethyl acetal (4.86 mL, 31.60 mmol) was added
dropwise. The mixture was refluxed for 4 h. After cooling, the
precipitate was filtered off and the solvent was evaporated in
vacuo. The crude residue was adsorbed on silica gel and purified by
flash chromatography eluting with hexane/EtOAc (4:1) to give 28
(2.60 g, 36%). .sup.1H NMR (500 MHz, CDCl.sub.3): .delta.: 10.53
(1H, s, CHO), 7.42 (1H, m, ArH), 7.14-7.12 (2H, m, ArH), 4.83 (1H,
t, J=5.30 Hz, CH), 4.21 (2H, d, J=5.35 Hz, CH.sub.2), 3.90 (3H, s,
OCH.sub.3), 3.74-3.71 (2H, m, CH.sub.2CH.sub.3), 3.60-3.57 (2H, m,
CH.sub.2CH.sub.3), 1.22 (6H, t, J=7.05 Hz, 2.times.CH.sub.3).
7-methoxybenzofuran-2-carbaldehyde (29)
[0209] A stirred solution of 28 (2.0 g, 7.46 mmol) in acetic acid
(10 mL) was refluxed for 24 h. After cooling, the solution was
evaporated to dryness. The crude product was adsorbed on silica gel
and purified by flash chromatography, eluting with hexane/EtOAc
(4:1) to give 29 (450 mg, 34%) as a white solid. .sup.1H NMR (500
MHz, CDCl.sub.3): .delta.: 9.89 (1H, s, CHO), 7.55 (1H, s, ArH),
7.30 (1H, d, J=6.95 Hz, ArH), 7.24 (1H, t, J=7.85, ArH), 6.97 (1H,
d, J=6.90 Hz), 4.02 (3H, s, OCH.sub.3).
2-(bromomethyl)-7-methoxybenzofuran (30)
[0210] Compound 29 (450 mg, 2.56 mmol) was dissolved in EtOH (10
mL). NaBH.sub.4 (104 mg, 2.81 mmol) was added portionwise at
0.degree. C., with vigorous stirring. The suspension was stirred at
0.degree. C. for 15 min and then at room temperature for 1.5 h.
Solvent was evaporated off in-vacuo. The resulting crude alcohol
residue was dissolved in toluene (5 mL) and the solution was cooled
to 0.degree. C. PBr.sub.3 (240 .mu.L, 2.56 mmol) was added dropwise
over 10 min. The reaction mixture was then brought up to room
temperature and stirred for 1 h. The solvent was evaporated off
in-vacuo. The residue was adsorbed on silica gel and purified by
flash chromatography, eluting with hexane/EtOAc (4:1) to give 30
(150 mg, 24%) as an oil. .sup.1H NMR (500 MHz, CDCl.sub.3):
.delta.: 7.19-7.17 (2H, m, ArH), 6.85 (1H, d, J=5.60, ArH), 6.78
(1H, s, ArH), 4.62 (2H, s, 2-CH.sub.2), 4.04 (3H, s,
OCH.sub.3).
7-((7-methoxybenzofuran-2-yl)methoxy)-4-methyl-2H-chromen-2-one
(31) VG029-05
[0211] Sodium ethoxide (12 mg, 0.18 mmol) was added to DMF (5 mL)
at 0.degree. C., and the suspension was stirred for 10 min.
7-hydroxy-4-methylcoumarin (32 mg, 0.17 mmol) was slowly added and
resulting mixture was stirred at this temp for 0.5 h. To this
mixture 30 (40 mg, 0.17 mmol) was added portionwise. Resulting
reaction mixture was stirred at room temperature for 2 h. DMF was
evaporated off in-vacuo and the residue was purified by flash
chromatography, eluting with hexane: EtOAc (3:1) to give 31 (14 mg,
25%) as a white solid m/z 337.04 (M+H), 673.13 (2M+H).
7-((5-bromobenzofuran-2-yl)methoxy)-4-methyl-2H-chromen-2-one (32)
VG035-04
##STR00037##
[0212]
7-((5-bromobenzofuran-2-yl)methoxy)-4-methyl-2H-chromen-2-one
(VG035-04)
[0213] Sodium ethoxide (76 mg, 1.10 mmol) was added to DMF (10 mL)
at 0.degree. C., and the suspension was stirred for 10 min.
7-hydroxy-4-methylcoumarin (215 mg, 1.22 mmol) was slowly added and
resulting mixture was stirred at this temp for 0.5 h. To this
mixture 5-bromo-2-(chloromethyl)benzofuran (250 mg, 1.02 mmol) was
added portionwise. Resulting reaction mixture was stirred at room
temperature for 2 h. DMF was evaporated off in-vacuo and the
residue was purified by flash chromatography, eluting with hexane:
EtOAc (3:1) to give 32 (120 mg, 31%) as a white solid. m/z 386
(M+H). H.sup.1 NMR (500 MHz, DMSO-d.sub.6): =7.91 (1H, d, J=2.0 Hz,
ArH), 7.71 (1H, d, J=8.80 Hz, ArH), 7.61 (1H, d, J=8.75, ArH), 7.49
(1H, dd, J=6.70 & 2.05 Hz, ArH), 7.20 (1H, d, J=2.45 Hz, ArH),
7.13 (1H, s, ArH), 7.09 (1H, dd, J=6.30 & 2.50, ArH), 6.25 (1H,
s, CH), 5.43 (2H, s, CH.sub.2), 2.41 (3H, s, CH.sub.3). .sup.13C
NMR CDEPT 135, (500 MHz, CDCl.sub.3): .delta.: 127.55 (ArCH),
126.59 (ArCH), 124.04 (ArCH), 113.32 (ArCH), 112.56 (ArCH), 111.44
(ArCH), 106.87 (ArCH), 101.66 (ArCH), 62.40 (CH.sub.2), 16.11
(CH.sub.3).
7-((5-chlorobenzofuran-2-yl)methoxy)-4-methyl-2H-chromen-2-one (36)
VG028-05
##STR00038##
[0214] 5-chloro-2-(2,2-diethoxyethoxy)benzaldehyde (33)
[0215] To a stirred suspension of 5-chloro-2-hydroxybenzaldehyde
(5.0 g, 32.1 mmol) and K.sub.2CO.sub.3 (4.87 g, 35.3 mmol) in DMF
(20 mL), bromoacetaldehyde diethyl acetal (5.43 mL, 35.3 mmol) was
added dropwise. The mixture was refluxed for 4 h. After cooling,
the precipitate was filtered off and the solvent was evaporated in
vacuo. The crude residue was adsorbed on silica gel and purified by
flash chromatography. The product was eluted with hexane/EtOAc
(4:1) to give 33 (4.10 g, 38%) as an oil. .sup.1H NMR (500 MHz,
CDCl.sub.3): .delta.: 10.42 (1H, s, CHO), 7.76 (1H, d, J=2.8 Hz,
ArH), 7.46 (1H, dd, J=6.15 & 2.75 Hz, ArH), 6.96 (1H, d, J=8.90
Hz, ArH), 4.87 (1H, t, J=5.25 Hz, CH), 4.10 (2H, d, J=5.25 Hz,
CH.sub.2), 3.80-3.77 (2H, m, CH.sub.2CH.sub.3), 3.67-3.62 (2H, m,
CH.sub.2CH.sub.3), 1.24 (6H, t, J=7.05 Hz, 2.times.CH.sub.3).
5-chlorobenzofuran-2-carbaldehyde (34)
[0216] A stirred solution of 33 (4.10 g, 15.07 mmol) in acetic acid
(20 mL) was refluxed for 24 h. After cooling, the solution was
evaporated to dryness. The crude product was adsorbed on silica gel
and purified by flash chromatography, eluting with hexane/EtOAc
(4:1) to give 34 (550 mg, 20%) as a white solid. .sup.1H NMR (500
MHz, CDCl.sub.3): .delta.: 9.91 (1H, s, CHO), 7.76 (1H, d, J=1.85
Hz, ArH), 7.57 (1H, d, J=8.90 Hz, ArH), 7.53 (1H, s, ArH), 7.50
(1H, dd, J=8.90 & 2.10 Hz, ArH).
2-(bromomethyl)-5-chlorobenzofuran (35)
[0217] Compound 34 (160 mg, 0.89 mmol) was dissolved in EtOH (5
mL). NaBH.sub.4 (36 mg, 0.98 mmol) was added portionwise at
0.degree. C., with vigorous stirring. The suspension was stirred at
0.degree. C. for 15 min and then at room temperature for 1.5 h.
Solvent was evaporated off in-vacuo. The resulting crude alcohol
residue was dissolved in toluene (5 mL) and the solution was cooled
to 0.degree. C. PBr.sub.3 (92 .mu.L, 0.98 mmol) was added dropwise
over 10 min. The mixture was then brought up to room temperature
and stirred for 1 h. The solvent was evaporated off in-vacuo. The
residue was adsorbed on silica gel and purified by flash
chromatography, eluting with hexane/EtOAc (4:1) to give 35 (128 mg,
57%) as an oil. .sup.1H NMR (500 MHz, CDCl.sub.3): .delta.: 7.48
(1H, d, J=2.05 Hz, ArH), 7.37 (1H, d, J=8.70, ArH), 7.25 (1H, dd,
J=8.80 & 2.05 Hz ArH), 6.68 (1H, s, ArH), 4.55 (2H, s,
2-CH.sub.2).
7-((5-chlorobenzofuran-2-yl)methoxy)-4-methyl-2H-chromen-2-one (36)
VG028-05
[0218] Sodium ethoxide (60 mg, 0.24 mmol) was added to DMF (5 mL)
at 0.degree. C., and the suspension was stirred for 10 min.
7-hydroxy-4-methylcoumarin (47 mg, 0.27 mmol) was slowly added and
resulting mixture was stirred at this temp for 0.5 h. To this
mixture 2-(bromomethyl)-5-methoxybenzofuran (40 mg, 0.17 mmol) was
added portionwise. Resulting reaction mixture was stirred at room
temperature for 2 h. DMF was evaporated off in-vacuo and the
residue was purified by flash chromatography, eluting with hexane:
EtOAc (3:1) to give the target compound as a white solid (2.7 mg,
3%). m/z 341.10 (M+H).
7-((5,7-dimethoxybenzofuran-2-yl)methoxy)-4-methyl-2H-chromen-2-one
(42) VG035-05
##STR00039##
[0219] 2-Bromo-3,5-dimethoxybenzaldehyde (37)
[0220] 3,5-dimethoxybenzaldehyde (12.6 g, 76 mmol) was dissolved in
acetic acid (350 mL). The resulting colourless solution was cooled
to 0.degree. C. A solution of bromine (3.9 mL) in acetic acid (50
mL) was added dropwise over 1 h. Once the addition was complete the
ice bath was removed and the resulting pale green solution was
stirred overnight at room temperature. Cold water was added to the
solution. The resulting white solid was collected by vacuum
filteration and rinsed with water. The solid was then redissolved
in EtOAc and adsorbed on silica gel. The product was purified by
flash chromatography, eluting with hexane/EtOAc (4:1) to give 37
(12.5 g, 66%) as a white solid. m/z=344.98 (M+H). .sup.1H NMR (500
MHz, CDCl.sub.3): .delta.: 10.43 (1H, s, CHO), 7.06 (1H, s, ArH),
6.73 (1H, s, ArH), 3.93 (3H, s, CH.sub.3O), 3.86 (3H, s,
CH.sub.3O). .sup.13C NMR (500 MHz, CDCl.sub.3): .delta.: 192.09
(CHO), 159.92 (C-5), 157.02 (C-3), 134.67 (C-1), 109.12 (C-2),
105.83, 103.37 (C-4 & C-6), 56.60 (OMe), 55.82 (OMe).
2-Hydroxy-3,5-dimethoxybenzaldehyde (38)
[0221] Morpholine (2.05 g, 24 mmol) and THF (40 mL) were placed in
a dry, three-necked, round bottomed flask equipped with a stirring
bar, septum cap, dropping funnel, thermometer, and argon inlet. The
flask was cooled in a dry ice-acetone bath to -50.degree. C., and a
solution of n-BuLi in hexane (1.6M, 15 mL, 24 mmol) was added all
at once. After 10 min a solution of the
2-bromo-3,5-dimethoxybenzadehyde 37 (4.9 g, 20 mmol) in THF (30 mL)
was added dropwise via a syringe over a period of 4 min, and the
mixture was cooled to .about.-75.degree. C. over 20 min. n-BuLi in
hexane (1.6M, 20 mL, 32 mmol) was then added dropwise over 45 min,
keeping the temperature at -75.degree. C. After complete addition
of n-BuLi the solution was stritted for 35 min. A solution of
nitrophenol (6.90 g, 46 mmol) in 10 mL THF was added from the
dropping funnel, keeping the temperature at -75.degree. C. The
resulting dark mixture was stirred at -75.degree. C. for 4 h and
then allowed to warm to room temperature. It was acidified to pH 1
with 6N HCl and stirred for 15 min. After dilution with brine (100
mL), THF was removed in-vacuo. The aqueous solution was extracted
with diethyl ether (4.times.40 mL). The combined organic layers
were extracted with 2 N NaOH (3.times.40 mL). The combined NaOH
extracts were washed with diethyl ether (3.times.20 mL) and then
acidified to pH 1 with concentrated HCl. The resulting mixture was
extracted with CH.sub.2Cl.sub.2 (3.times.20 mL), and the combined
organic extracts were washed with brine, dried (MgSO.sub.4) and
adsorbed on silica gel. The product was purified by flash
chromatography, eluting with EtOAc/hexane (1:2) to give 38 (2.0 g,
55%) as a yellow solid. m/z=183.06 (M+H). .sup.1H NMR (500 MHz,
CDCl.sub.3): .delta.: 10.71 (1H, s, OH), 9.91 (1H, s, CHO), 6.77
(1H, d, J.sub.4, 6=2.8 Hz, H-6), 6.61 (1H, d, J.sub.4, 6=2.8 Hz,
H-4), 3.92 (3H, s, OMe), 3.84 (3H, s, OMe). .sup.13C NMR CDEPT135
(500 MHz, CDCl.sub.3): .delta.: 196.11 (CHO), 107.93 (C-6), 103.90
(C-4), 56.29 (OMe), 55.83 (OMe).
2-(2,2-Diethoxyethoxy)-3,5-dimethoxybenzaldehyde (39)
[0222] To a stirred suspension containing 38 (1.1 g, 6.0 mmol) and
K.sub.2CO.sub.3 (1.0 g, 7.2 mmol) in DMF (100 mL),
bromoacetaldehyde diethyl acetal (0.93 mL, 6.0 mmol) was added
dropwise. The mixture was refluxed for 4 h. After cooling, the
precipitate was filtered off and the solvent was evaporated in
vacuo. The crude residue was adsorbed on silica gel and purified by
flash chromatography. The product was eluted with hexane/EtOAc
(4:1) to give 39 (1.2 g, 67%) as an oil. .sup.1H NMR (500 MHz,
CDCl.sub.3): .delta.: 10.50 (1H, s, CHO), 6.88 (1H, d, J.sub.4,
6=2.9 Hz, H-6), 6.74 (1H, d, J.sub.4, 6=2.9 Hz, H-4), 4.83 (1H, t,
J.sub.4, 6=5.3 Hz, CH), 4.14 (2H, d, J.sub.4, 6=5.3 Hz, CH.sub.2),
3.88 (3H, s, OMe), 3.83 (3H, s, OMe), 3.77-3.71) (2H, m,
CH.sub.2CH.sub.3), 3.63-3.58 (2H, m, CH.sub.2CH.sub.3), 1.24 (6H,
t, J=7.1 Hz, 2.times.CH.sub.3).
5,7-dimethoxybenzofuran-2-carbaldehyde (40)
[0223] A stirred solution of 39 (1.2 g, 4.0 mmol) in acetic acid
(35 mL) was refluxed for 16 h. After cooling, the solution was
evaporated to dryness. The crude product was adsorbed on silica gel
and purified by flash chromatography, eluting with hexane/EtOAc
(2:1) to give 40 (230 mg, 28%) as a white solid. .sup.1H NMR (500
MHz, CDCl.sub.3): .delta.: 9.89 (1H, s, CHO), 7.50 (1H, s, H-3),
6.69 (1H, d, J.sub.4, 6=2.2 Hz, H-6), 6.64 (1H, d, J.sub.4, 6=2.2
Hz, H-4), 4.01 (3H, s, OMe), 3.87 (3H, s, OMe). .sup.13C NMR CDEPT
135, (500 MHz, CDCl.sub.3): .delta.: 179.91 (CHO), 153.37 (C-2),
116.10 (C-3), 101.80 (C-6), 94.90 (C-4), 56.20 (OMe), 55.88
(OMe).
(5,7-dimethoxybenzofuran-2-yl)methanol (41)
[0224] Compound 39 (460 mg, 2.23 mmol) was dissolved in THF (5 mL)
and EtOH (1 mL). NaBH.sub.4 (102 mg, 2.68 mmol) was added
portionwise at 0.degree. C., with vigorous stirring. The suspension
was stirred at 0.degree. C. for 15 min and then at room temperature
for 1 h. Solvents were evaporated off in-vacuo. The crude residue
was taken up in EtOAc and washed with water, brine and dried
(MgSO.sub.4). The residue was adsorbed on silica gel and purified
by flash chromatography, eluting with hexane/EtOAc (1:1) to give 41
(388 mg, 82%) as a white solid. m/z=209.08 (M+H). .sup.1H NMR (500
MHz, CDCl.sub.3): .delta.: 6.62 (1H, s, H-3), 6.60 (1H, s, H-6),
6.46 (1H, s, H-4), 4.76 (2H, s, 2-CH.sub.2), 3.99 (3H, s, OMe),
3.85 (3H, s, OMe). .sup.13C NMR (500 MHz, CDCl.sub.3): .delta.:
157.23 (C-5), 156.72 (C-1), 145.36 (C-7), 139.50 (C-1a), 129.38
(C-4a), 104.62 (C-3), 96.96 (C-6), 94.58 (C-4), 57.98 (2-CH.sub.2),
55.95 (OMe), 55.83 (OMe).
7-((5,7-dimethoxybenzofuran-2-yl)methoxy)-4-methyl-2H-chromen-2-one
(42) VG035-05
[0225] Compound 41 (130 mg, 0.63 mmol) was dissolved in toluene (5
mL) and the solution was cooled to 0.degree. C. PBr.sub.3 (64
.mu.L, 0.69 mmol) was added dropwise over 10 min. The reaction
mixture was then brought up to room temperature and stirred for 1
h. The solvent was evaporated off in-vacuo. The crude residue was
used in the next step. Sodium ethoxide (80 mg, 0.24 mmol) was added
to DMF (5 mL) at 0.degree. C., and the suspension was stirred for
10 min. 7-hydroxy-4-methylcoumarin (47 mg, 0.27 mmol) was slowly
added and resulting mixture was stirred at this temp for 0.5 h. To
this mixture 2-(bromomethyl)-5-methoxybenzofuran (40 mg, 0.17 mmol)
was added portionwise. Resulting reaction mixture was stirred at
room temperature for 2 h. DMF was evaporated off in-vacuo and the
residue was purified by flash chromatography, eluting with hexane:
EtOAc (3:1) to give 42 (2.7 mg, 3%) as a white solid. m/z=367.05
(M+H), 733.15 (2M+H).
4-methyl-7-(naphthalene-1-ylmethoxy)-2H-chromen-2-one (43)
TLE-M1-SU001A
##STR00040##
[0227] 1-Naphthalene methanol (2.0 g, 12.7 mmol) was dissolved in
toluene (30 mL) and pyridine (1.02 mL, 12.7 mmol) was added. The
solution was cooled to 0.degree. C. PBr.sub.3 (1.19 mL, 12.7 mmol)
was added dropwise over 15 min. The reaction mixture was then
brought up to room temperature and stirred for 1 h. The mixture was
washed with K.sub.2CO.sub.3 solution and extracted with EtOAc
(3.times.30 mL). The EtOAc layer was washed with brine and dried
(MgSO.sub.4). The solvent was evaporated off in-vacuo to give
1-(bromomethyl)naphthalene (1.5 g, 53%) as a colourless oil, This
intermediate was used in the following reaction.
[0228] Sodium ethoxide (169 mg, 2.49 mmol) was added to DMF (5 mL)
at 0.degree. C., and the suspension was stirred for 10 min.
7-hydroxy-4-methylcoumarin (438 mg, 2.49 mmol) was slowly added and
resulting mixture was stirred at this temp for 0.5 h, then allowed
to reach room temperature. To this mixture
1-(bromomethyl)naphthalene (500 mg, 2.26 mmol) was added
portionwise. Resulting reaction mixture was stirred at room
temperature for 16 h. DMF was evaporated off in-vacuo and the
residue was taken up in EtOAc, and washed with brine (2.times.50
mL), water (2.times.50 mL) and 1M NaOH (2.times.30 mL). The organic
layer was dried (MgSO.sub.4) and product was purified by flash
chromatography, eluting with hexane:EtOAc (2:1) to give 43 (200 mg,
28%) as a white solid. Mpt=181-183.degree. C. H.sup.1 NMR (500 MHz,
acetone-d.sub.6): .delta.=8.10 (1H, d, ArH), 8.00-7.99 (2H, m, Ar),
7.97-7.71 (2H, m, ArH), 7.70-7.53 (3H, m, ArH), 7.24 (1H, s, ArH),
7.09 (1H, d, CH), 6.23 (1H, s, CH), 5.68 (2H, s, CH.sub.2), 2.40
(3H, s, CH.sub.3).
4-methyl-7-(naphthalen-2-ylmethoxy)-2H-chromen-2-one (44)
VG040-03
##STR00041##
[0230] Naphthalen-2-ylmethanol (2.0 g, 12.7 mmol) was dissolved in
toluene (30 mL) and pyridine (1.02 mL, 12.7 mmol) was added. The
solution was cooled to 0.degree. C. PBr.sub.3 (1.19 mL, 12.7 mmol)
was added dropwise over 15 min. The mixture was then brought up to
room temperature and stirred for 1 h. The mixture was washed with
K.sub.2CO.sub.3 solution and extracted with EtOAc (3.times.30 mL).
The EtOAc layer was washed with brine and dried (MgSO.sub.4). The
solvent was evaporated off in-vacuo to give crude
1-(bromomethyl)naphthalene. This intermediate was used in the
following reactions.
[0231] Sodium ethoxide (169 mg, 2.49 mmol) was added to DMF (5 mL)
at 0.degree. C., and the suspension was stirred for 10 min.
7-hydroxy-4-methylcoumarin (438 mg, 2.49 mmol) was slowly added and
resulting mixture was stirred at this temp for 0.5 h, then allowed
to reach room temp. To this mixture 1-(bromomethyl)naphthalene (500
mg, 2.26 mmol) was added portionwise. Resulting reaction mixture
was stirred at room temperature for 16 h. DMF was evaporated off
in-vacuo and the residue was taken up in EtOAc, and washed with
brine (2.times.50 mL), water (2.times.50 mL) and 1M NaOH
(2.times.30 mL). The organic layer was dried (MgSO.sub.4) and
product was purified by flash chromatography, eluting with hexane:
EtOAc (2:1) to give 44 (1.44 g, 36%) as a white solid. m/z=317.12
(M+H), 633.24 (2M+H). H.sup.1 NMR (500 MHz, CDCl.sub.3):
.delta.=7.93-7.87 (4H, m, ArH), 7.58-7.52 (4H, m, Ar), 6.97 (1H, t,
J=2.43 Hz, ArH), 6.16 (1H, s, ArH), 5.32 (2H, s, CH.sub.2), 2.41
(3H, s, CH.sub.3).
7-(benzhydryloxy)-4-methyl-2H-chromen-2-one (45) TLE-M1-SU004A
##STR00042##
[0233] Sodium ethoxide (165 mg, 2.43 mmol) was added to DMF at
0.degree. C., and the suspension was stirred for 10 min.
7-hydroxy-4-methylcoumarin (428 mg, 2.43 mmol) was slowly added and
resulting mixture was stirred at this temp for 0.5 h, then allowed
to reach room temp. To this mixture diphenylmethyl bromide (500 mg,
2.02 mmol) was added portionwise. Resulting reaction mixture was
stirred at room temperature for 16 h. DMF was evaporated off
in-vacuo and the residue was taken up in EtOAc, and washed with
brine (2.times.30 mL), water (2.times.30 mL) and 1M NaOH
(2.times.30 mL). The organic layer was dried (MgSO.sub.4) and
product was purified by flash chromatography, eluting with hexane:
EtOAc (2:1) to give 45 (200 mg, 29%) as a white solid.
Mpt=146-148.degree. C. H.sup.1 NMR (500 MHz, DMSO-d.sub.6):
.delta.=7.63 (1H, d, ArH), 7.53 (4H, d, ArH), 7.38 (4H, t, ArH),
7.29 (2H, t, ArH), 7.09 (2H, d, ArH), 7.04 (1H, d, ArH), 6.75 (1H,
s, ArH), 6.18 (1H, s, CH), 2.37 (3H, s, CH.sub.3). .sup.13C NMR
(500 MHz, DMSO-d.sub.6, DEPT135): .delta.=160.2, 154.4, 153.2,
140.8, 129.90, 128.0, 126.8, 113.7, 111.4, 111.2, 102.9, 80.2,
18.2.
4-methyl-7-(1-(naphthalen-2-yl)ethoxy)-2H-chromen-2-one (46)
VG039-03
##STR00043##
[0235] 1-(Naphthalen-2-yl)ethanol (2.0 g, 11.6 mmol) was dissolved
in toluene (30 mL). The solution was cooled to 0.degree. C.
PBr.sub.3 (1.09 mL, 11.6 mmol) was added dropwise over 15 min. The
reaction mixture was then brought up to room temperature and
stirred for 1 h. The mixture was washed with K.sub.2CO.sub.3
solution and extracted with EtOAc (3.times.30 mL). The EtOAc layer
was washed with brine and dried (MgSO.sub.4). The solvent was
evaporated off in-vacuo to give crude 2-(1-bromoethyl)naphthalene.
This intermediate was used in the following step.
[0236] Sodium ethoxide (63.4 mg, 0.93 mmol) was added to DMF (5 mL)
at 0.degree. C., and the suspension was stirred for 10 min.
7-hydroxy-4-methylcoumarin (147 mg, 0.84 mmol) was slowly added and
resulting mixture was stirred at this temperature for 0.5 h, then
allowed to reach room temperature. To this mixture
2-(1-bromoethyl)naphthalene (200 mg, 0.85 mmol) was added
portionwise. Resulting reaction mixture was stirred at room
temperature for 16 h. DMF was evaporated off in-vacuo and the
residue was taken up in EtOAc, and washed with brine (2.times.50
mL), water (2.times.50 mL) and 1M NaOH (2.times.30 mL). The organic
layer was dried (MgSO.sub.4) and product was purified by flash
chromatography, eluting with hexane: EtOAc (2:1) to give 46 (60 mg,
21%) as a white solid. m/z=331.15 (M+H), 661.29 (2M+H). H.sup.1 NMR
(500 MHz, DMSO-d.sub.6): .delta.=7.97 (1H, s, ArH), 7.93-7.86 (3H,
m, ArH), 7.60-7.50 (4H, m, Ar), 6.99 (1H, q, J=6.45 & 2.35 Hz,
ArH), 6.96 (1H, d, J=2.40 Hz, ArH), 6.13 (1H, s, ArH), 5.84 (1H, q,
J=6.35 Hz, CH), 2.26 (3H, s, CH.sub.3), 1.67 (3H, d, J=6.35 Hz,
CH.sub.3).
7-(anthracen-9-ylmethoxy)-4-methyl-2H-chromen-2-one (48)
SU06-02
##STR00044##
[0237] 9-(bromomethyl)anthracene (47)
[0238] To a stirring suspension of 9-anthracenemethanol (2.0 g, 9.6
mmol) at 0.degree. C. in toluene (100 ml) was added PBr.sub.3 (1.2
mL, 12.51 mmol) and the suspension was stirred at 0.degree. C. for
1 h. The reaction mixture was then brought up to room temperature
and let to stir for further 1 h. The mixture turned into a yellow
solution. K.sub.2CO.sub.3 (10 mL) was added to quench the reaction.
Toluene was evaporated off in-vacuo. The residue was taken up in
EtOAc and washed with saturated aqueous K.sub.2CO.sub.3, water and
brine and dried (MgSO.sub.4). The solvent was evaporated off
in-vacuo and the crude residue was purified by flash
chromatography, eluting with hexane:EtOAc (2:1) to give 47 (1.4 g,
54%) as yellow solid. H.sup.1 NMR (500 MHz, CDCl.sub.3):
.delta.=8.45 (1H, s, Ar-10H), 8.27 (2H, d, Ar-1,8 H), 8.00 (2H, d,
Ar-4, 6H), 7.62 (2H, d, Ar-2, 7H), 7.48 (2H, d, Ar-3, H), 5.50 (2H,
s, CH.sub.2).
7-(anthracen-9-ylmethoxy)-4-methyl-2H-chromen-2-one (48)
SU06-02
[0239] Sodium ethoxide (151 mg, 2.21 mmol) was added to DMF (5 mL)
at 0.degree. C., and the suspension was stirred for 10 min.
7-hydroxy-4-methylcoumarin (390 mg, 2.21 mmol) was slowly added and
resulting mixture was stirred at this temp for 0.5 h, then allowed
to reach room temp. To this mixture 47 (500 mg, 1.85 mmol) was
added portionwise. Resulting reaction mixture was stirred at room
temperature for 16 h. DMF was evaporated off in-vacuo and the
residue was taken up in EtOAc, and washed with brine, water and 1M
NaOH (2.times.30 mL). The organic layer was dried (MgSO.sub.4) and
product was purified by flash chromatography, eluting with hexane:
EtOAc (2:1) to give 48 (200 mg, 30%) as a yellow solid.
Mpt=216-218.degree. C. H.sup.1 NMR (500 MHz, DMSO-d.sub.6):
.delta.=8.10 (1H, d, ArH), 8.00-7.99 (2H, m, Ar), 7.97-7.71 (2H, m,
ArH), 7.70-7.53 (3H, m, ArH), 7.24 (1H, s, ArH), 7.09 (1H, d, CH),
6.23 (1H, s, CH), 5.68 (2H, s, CH.sub.2), 2.40 (3H, s, CH.sub.3).
.sup.13C NMR (500 MHz, DMSO-d.sub.6, DEPT135): .delta.=160.8 (qC),
152.0 (2.times.qC), 129.0 (2.times.CH), 128.9 (2.times.CH), 126.8,
(2.times.CH), 126.8 (Ar CH), 126.5 (Ar CH), 125.3 (Ar CH), 124.1
(Ar CH), 112.9 (coumarin 3-CH), 111.2 (coumarin 6-CH), 101.7
(coumarin 8-CH), 62.9 (CH.sub.2), 18.2 (CH.sub.3).
7-(bis(4-methoxyphenyl)methoxy)-4-methyl-2H-chromen-2-one (49)
SU010-02
##STR00045##
[0241] Bis(4-methoxyphenyl)methanol (2.0 g, 8.2 mmol) was dissolved
in toluene (60 mL) and pyridine (661 .mu.L, 8.2 mmol)) was added.
The solution was cooled to 0.degree. C. PBr.sub.3 (768 .mu.L, 8.2
mmol) was added dropwise over 15 min. The reaction mixture was
warmed to room temperature and stirred for 1 h. The mixture was
washed with K.sub.2CO.sub.3 solution and extracted with EtOAc
(3.times.30 mL). The EtOAc layer was washed with brine and dried
(MgSO.sub.4). The solvent was evaporated off in-vacuo to give the
crude product 4,4'-(bromomethylene)bis(methoxybenzene) (780 mg,
31%), as a colourless oil. This was used in the next reaction step
without further purification. Sodium ethoxide (133 mg, 1.96 mmol)
was added to DMF (5 mL) at 0.degree. C., and the suspension was
stirred for 10 min. 7-hydroxy-4-methylcoumarin (345 mg, 1.96 mmol)
was slowly added and resulting mixture was stirred at this temp for
0.5 h. To this mixture 4,4'-(bromomethylene)bis(methoxybenzene (500
mg, 1.63 mmol) was added portionwise. Resulting reaction mixture
was stirred at room temperature for 16 h. DMF was evaporated off
in-vacuo and the residue was taken up in EtOAc, and washed with
brine, water and 1M NaOH (2.times.30 mL). The organic layer was
dried (MgSO.sub.4) and product was purified by flash
chromatography, eluting with hexane: EtOAc (2:1) to give 49 (100
mg, 15%) as a white solid. Mpt=142-145.degree. C. m/z=403 (M+H).
H.sup.1 NMR (500 MHz, acetone-d.sub.6): .delta.=7.60 (1H, d, ArH),
7.45 (4H, d, ArH), 7.04 (1H, d, ArH), 6.94 (5H, d, ArH), 6.56 (1H,
s ArH), 6.10 (1H, s, qCH), 3.78 (6H, s, 2.times.CH.sub.3O), 2.39
(3H, s, CH.sub.3). .sup.13C NMR (500 MHz, acetone-d.sub.6,
DEPT135): .delta.=206.3 (qC), 134.1 (2.times.qC), 129.3
(2.times.CH), 129.2 (2.times.CH), 129.0, (2.times.CH), 126.9 (Ar
CH), 114.4 (Ar CH), 114.7 (Ar CH), 115.1 (Ar CH), 112.5 (Ar CH),
104.0 (Ar CH), 81.7 (CH), 55.6 (2.times.CH.sub.3), 18.2
(CH.sub.3).
7-((1H-benzo[d]imidazol-2-yl)methoxy)-4-methyl-2H-chromen-2-one
(50) VG033-03
##STR00046##
[0243] Sodium ethoxide (82 mg, 1.20 mmol) was added to DMF (10 mL)
at 0.degree. C., and the suspension was stirred for 10 min.
7-hydroxy-4-methylcoumarin (253 mg, 1.44 mmol) was slowly added and
resulting mixture was stirred at this temp for 0.5 h. To this
mixture 2-(chloromethyl)-1H-benzo[d]imidazole (200 mg, 1.20 mmol)
was added portionwise. Resulting reaction mixture was stirred at
room temp for 2 h. DMF was evaporated off in-vacuo and the residue
was purified by flash chromatography, eluting with hexane:EtOAc
(3:1) to give 50 (200 mg, 54%) as a white solid. m/z=307.11 (M+H),
613.22 (2M+H). H.sup.1 NMR (500 MHz, DMSO-d.sub.6): .delta.=12.75
(1H, brs, NH), 7.74 (1H, d, J=8.85 Hz, ArH), 7.60-7.59 (2H, m,
ArH), 7.23-7.12 (4H, m, ArH), 6.25 (1H, s, ArH), 5.48 (2H, s,
CH.sub.2), 2.40 (3H, s, CH.sub.3). .sup.13C NMR CDEPT 135, (500
MHz, CDCl.sub.3): .delta.: 206.52 (qC), 160.80, 160.03, 154.52,
153.33, 149.27, 126.57, 126.28, 122.04, 119.42, 113.64, 112.50,
111.47, 101.86, 101.77, 64.23, 30.67, 18.10.
7-(benzo[d]thiazol-2-ylmethoxy)-4-methyl-2H-chromen-2-one (51)
VG014-04
##STR00047##
[0245] Sodium ethoxide (30 mg, 0.44 mmol) was added to DMF (10 mL)
at 0.degree. C., and the suspension was stirred for 10 min.
7-hydroxy-4-methylcoumarin (77 mg, 0.44 mmol) was slowly added and
resulting mixture was stirred at this temp for 0.5 h. To this
mixture 2-(chloromethyl)-1H-benzo[d]imidazole (100 mg, 0.44 mmol)
was added portionwise. Resulting reaction mixture was stirred at
room temperature for 2 h. DMF was evaporated off in-vacuo and the
residue was purified by flash chromatography, eluting with hexane:
EtOAc (3:1) to give 51 (25 mg, 18%) as a white solid. m/z=324.06
(M+H), 647.12 (2M+H).
4-methyl-7-(4-(thiophen-2-yl)benzyloxy)-2H-chromen-2-one (52)
VG015-04
##STR00048##
[0247] Sodium ethoxide (27 mg, 0.40 mmol) was added to DMF (10 mL)
at 0.degree. C., and the suspension was stirred for 10 min.
7-hydroxy-4-methylcoumarin (70 mg, 0.40 mmol) was slowly added and
resulting mixture was stirred at this temp for 0.5 h. To this
mixture 2-(chloromethyl)-1H-benzo[d]imidazole (100 mg, 0.40 mmol)
was added portionwise. Resulting reaction mixture was stirred at
room temp for 2 h. DMF was evaporated off in-vacuo and the residue
was purified by flash chromatography, eluting with hexane: EtOAc
(3:1) to give 52 (30 mg, 18%) as a white solid. m/z=349.09 (M+H),
697.16 (2M+H).
6-(benzhydrylthio)-9H-purine (53) (VG015-02)
##STR00049##
[0249] 6-Mercaptopurine (151 mg, 0.88 mmol) was dissolved in DMF (5
mL). K.sub.2CO.sub.3 (122 mg, 1.2 mmol) was added and to the
resulting suspension, diphenyl methylbromide (200 mg, 0.8 mmol) was
added. The resulting reaction mixture was stirred at room
temperature for 4 h. The mixture was poured on ice and the
resulting precipitate was separated by filteration, washed with
ether and dried in vacuo to give 53 (35 mg, 14%) as a white solid.
m/z=319 (M+H). H.sup.1 NMR (500 MHz, acetone): .delta.=8.46 (1H, s,
CH), 8.2 (1H, s, CH), 7.4 (4H, m, CH, J=3), 7.2 (4H, m, CH,
J=3.83), 7.1 (2H, m, CH, J=2.12), 6.7 (1H, s, CH).
3. Carbamate-Linked Nucleoside Analogue Prodrugs
TABLE-US-00003 [0250] SU001- 03 ##STR00050## SU0044- 2a/02
(.dagger.) ##STR00051## SU0023/ 02 ##STR00052## SU0044- 3a/02
(.dagger.) ##STR00053## SU050- 03 ##STR00054## SU048- 04
##STR00055##
naphthalen-1-ylmethyl
1-(3,4-dihydroxy-5-(hydroxymethyl)tetrahydrofuran-2-yl)-2-oxo-1,2-dihydro-
pyrimidin-4-ylcarbamate (55) SU001-03
##STR00056##
[0252] Naphthalen-1-ylmethanol (3.0 g, 19.0 mmol) was added in one
portion to COCl.sub.2 (13.3 mL, as 20% solution of COCl.sub.2 in
toluene) in THF (30 mL). The reaction was stirred at room
temperature for 2 h. Excess COCl.sub.2 and THE was removed under
reduced pressure. The solid residue was dissolved in hot hexane and
filtered. The hexane was then slowly evaporated off in vacuo to
obtain the chloroformate intermediate 54 as a white solid. This was
used straight away in the following step. 54 (330 mg, 1.5 mmol) and
KHCO.sub.3 (252 mg, 2.52 mmol) were added to a solution of
cytarabine.HCl (243 mg, 0.87 mmol) in dimethyl acetamide (5 mL),
and the mixture was stirred for 16 h at room temperature. The
solvent was evaporated off in vacuo and the product was purified by
flash chromatography, eluting with a gradient of 2.5%-12% MeOH in
DCM to obtain 55 (38 mg, 10%) as a white solid. m/z=428.15
(M+H).
naphthalen-1-ylmethyl
1-(3,3-difluoro-4-hydroxy-5-(hydroxymethyl)tetrahydrofuran-2-yl)-2-oxo-1,-
2-dihydropyrimidin-4-ylcarbamate (56) SU0023-02
##STR00057##
[0254] Naphthalen-1-ylmethanol (1.0 g, 6.3 mmol) was added in one
portion to COCl.sub.2 (4.4 mL, as 20% solution of COCl.sub.2 in
toluene) in THF (20 mL). The reaction was stirred at room
temperature for 2 h. Excess COCl.sub.2 and THF was removed under
reduced pressure. The solid residue was dissolved in hot hexane and
filtered. The hexane solvent was then slowly evaporated off in
vacuo to obtain the chloroformate intermediate 54 as a white solid.
This was used straight away in the following step. Gemcitabine.HCl
(200 mg, 0.67 mmol) was dissolved in H.sub.2O (2 mL). To this was
added KHCO.sub.3 (67 mg, 0.67 mmol) and 54 (147 mg, 0.67 mmol),
predissolved in ethyl acetate (5 mL). The mixture was stirred at
100.degree. C. for 16 h. The solvent was evaporated off in vacuo
and the product was purified by flash chromatography, eluting with
3% MeOH in ethyl acetate to obtain 56 (15 mg, 5%) as an oil.
m/z=448.13 (M+H).
benzyl
1-(3,3-difluoro-4-hydroxy-5-(hydroxymethyl)tetrahydrofuran-2-yl)-2--
oxo-1,2-dihydropyrimidin-4-ylcarbamate (57) SU0044-2a/02
##STR00058##
[0256] Gemcitabine.HCl (200 mg, 0.67 mmol) was dissolved in
H.sub.2O (2 mL). To this was added KHCO.sub.3 (67 mg, 0.67 mmol)
and benzyl carbonochloridate (95 .mu.L, 0.67 mmol), predissolved in
ethyl acetate (5 mL). The mixture was stirred at 80.degree. C. for
16 h. The solvent was evaporated off in vacuo and the product was
purified by flash chromatography, eluting with 3% MeOH in ethyl
acetate to give 57 (40 mg, 15%) as an oil. m/z=398.12 (M+H).
benzyl
1-(3,4-dihydroxy-5-(hydroxymethyl)tetrahydrofuran-2-yl)-2-oxo-1,2-d-
ihydropyrimidin-4-ylcarbamate (58) SU0044-3a/02
##STR00059##
[0258] Cytarabine.HCl (200 mg, 0.72 mmol) was dissolved in H.sub.2O
(2 mL). To this was added KHCO.sub.3 (72 mg, 0.72 mmol) and benzyl
carbonochloridate (107 .mu.L, 0.72 mmol), predissolved in ethyl
acetate (5 mL). The mixture was stirred at 80.degree. C. for 16 h.
The solvent was evaporated off in vacuo and the product was
purified by flash chromatography, eluting with 3% MeOH in ethyl
acetate to give 58 (40 mg, 15%) as an oil. m/z=378.13 (M+H).
benzofuran-2-ylmethyl 4-nitrophenyl carbonate (60) and
(5,7-dimethoxybenzofuran-2-yl)methyl 4-nitrophenyl carbonate
(61)
##STR00060##
[0259] Benzofuran-2-ylmethyl 4-nitrophenyl carbonate (60)
[0260] A solution of benzofuran-2-ylmethanol 59 (300 mg, 2.03 mmol)
in THF (5 mL) was cooled to 0.degree. C. TEA (280 .mu.L, 2.03 mmol)
was added dropwise followed by the portionwise addition of
p-nitrophenyl chloroformate (282 mg, 3.05 mmol). The resulting
solution was stirred at room temperature for 2 h. Solvent was
evaporated off in vacuo and the crude residue was purified by flash
chromatography, eluting with hexane: EtOAc (3:1) to give 60 (350
mg, 54%) as a white solid. H.sup.1 NMR (500 MHz, CDCl.sub.3):
.delta.=8.31 (2H, m, ArH), 7.62 (1H, d, J=7.70 Hz, ArH), 7.54 (1H,
d, J=7.70 Hz, ArH), 7.43-7.27 (3H, m, ArH), 7.29 (1H, t, J=7.72 Hz,
ArH), 6.93 (1H, s, ArH), 5.43 (2H, s, CH.sub.2). .sup.13C NMR CDEPT
135, (500 MHz, CDCl.sub.3): .delta.=125.47, 125.37, 123.23, 121.80,
121.66, 111.60, 108.53, 62.95.
(5,7-dimethoxybenzofuran-2-yl)methyl 4-nitrophenyl carbonate
(61)
[0261] A solution of (5,7-dimethoxybenzofuran-2-yl)methanol 6 (100
mg, 0.48 mmol) in THF (3 mL) was cooled to 0.degree. C. TEA (69 uL,
0.48 mmol) was added dropwise followed by the portionwise addition
of p-nitrophenyl chloroformate (100 mg, 0.72 mmol). The resulting
solution was stirred at room temperature for 2 h. Solvent was
evaporated off in vacuo and the crude residue was purified by flash
chromatography, eluting with hexane: EtOAc (3:1), to give 61 (120
mg, 67%) as a white solid. H.sup.1 NMR (500 MHz, CDCl.sub.3):
.delta.=8.29 (2H, d, J=9.0 Hz, ArH), 7.40 (2H, d, J=9.0 Hz, ArH),
6.82 (1H, s, ArH), 6.63 (1H, s, ArH), 6.52 (1H, s, ArH), 5.39 (2H,
s, CH.sub.2), 4.00 (3H, s, OCH.sub.3), 3.85 (3H, s, OCH.sub.3).
benzofuran-2-ylmethyl
1-(3,3-difluoro-4-hydroxy-5-(hydroxymethyl)tetrahydrofuran-2-yl)-2-oxo-1,-
2-dihydropyrimidin-4-ylcarbamate (65) SU050-03 and
(5,7-dimethoxybenzofuran-2-yl)methyl
1-(3,3-difluoro-4-hydroxy-5-(hydroxymethyl)-tetrahydrofuran-2-yl)-2-oxo-1-
,2-dihydropyrimidin-4-ylcarbamate (66) SU048-04
##STR00061##
[0262]
4-amino-1-(9,9-difluoro-2,2,4,4-tetraisopropyltetrahydro-6H-furo[3,-
2-f][1,3,5,2,4]trioxadisilocin-8-yl)pyrimidin-2(1H)-one (62)
[0263] Gemcitabine.HCl (1.0 g, 3.3 mmol), was stirred in pyridine
(10 mL) for 10 min (2.times.5 mL). The pyridine was evaporated off.
The pyridine (10 mL) was added and
1,1,3,3,-tetraisopropyldisiloxane (1.17 mL, 3.63 mmol) was added
dropwise. Resulting mixture was stirred at 100.degree. C. for 16 h.
A further portion of 1,1,3,3,-tetraisopropyldisiloxane (1 mL) was
added and the mixture was stirred at 120.degree. C. for 1 h.
Reaction mixture was cooled to room temperature and solvent was
evaporated off in vacuo. The resulting crude solid was
recrystalised from EtOAc/ether (1:1) to give 62 (600 mg, 36%) as a
white solid. m/z=506.23 (M+H).
benzofuran-2-ylmethyl
1-(9,9-difluoro-2,2,4,4-tetraisopropyltetrahydro-6H-furo[3,2-f][1,3,5,2,4-
]trioxadisilocin-8-yl)-2-oxo-1,2-dihydropyrimidin-4-ylcarbamate
(63)
[0264] To a stirred solution of 62 (300 mg, 0.59 mmol) in THF (5
mL) was added benzofuran-2-ylmethyl 4-nitrophenyl carbonate (223
mg, 0.71 mmol). The resulting solution was stirred at 100.degree.
C. for 4 days. Solvent was evaporated off in vacuo and the product
was purified by preparative HPLC to give 63 (350 mg, 87%) as an
oil. m/z=680.0 (M+H), 1359.49 (2M+H).
benzofuran-2-ylmethyl
1-(3,3-difluoro-4-hydroxy-5-(hydroxymethyl)tetrahydrofuran-2-yl)-2-oxo-1,-
2-dihydropyrimidin-4-ylcarbamate (65) SU050-03
[0265] Compound 63 (200 mg, 0.29 mmol) was dissolved in THF (1.5
mL). To this was added tetra-n-butylammonium fluoride and the
resulting solution was stirred at room temperature for 15 min.
Solvent was evaporated off in vacuo. The product was purified by
flash chromatography, eluting with 5% MeOH in EtOAc to give 65 (30
mg, 23%) as an oil. m/z=438.14 (M+H), 874.24 (2M+H).
(5,7-dimethoxybenzofuran-2-yl)methyl
1-(3,3-difluoro-4-hydroxy-5-(hydroxymethyl)-tetrahydrofuran-2-yl)-2-oxo-1-
,2-dihydropyrimidin-4-ylcarbamate (66) SU048-04
[0266] To a stirred solution of 62 (108 mg, 0.21 mmol) in THF (5
mL) was added 61 (100 mg, 0.27 mmol). The resulting solution was
stirred at 100.degree. C. for 4 days. Solvent was evaporated off in
vacuo to give 64 as an oil. This was used in the next step without
further purification. Compound 64 (100 mg, 0.14 mmol) was dissolved
in THF (1.5 mL). To this was added tetra-n-butylammonium fluoride
and the resulting solution was stirred at room temperature for 15
min. Solvent was evaporated off in-vacuo. The product was purified
by flash chromatography, eluting with 5% MeOH in EtOAc to give 66
(18 mg, 26%) as an oil. m/z=498.14 (M+H), 995.29 (2M+H). H.sup.1
NMR (500 MHz, acetone-d.sub.6): .delta.=9.60 (1H, bs, NH), 8.34
(1H, d, J=7.62 Hz, ArH), 7.26 (1H, d, J=9.00 Hz, ArH), 6.92 (1H, s,
ArH), 6.71 (1H. d, J=2.20 Hz, ArH), 6.56 (1H, d, J=2.20 Hz, ArH),
6.26 (1H, t, J=7.56 Hz, CH), 5.64 (2H, s, CH.sub.2), 4.55-4.45 (1H,
m, CH), 4.05-4.02 (2H, m, CH.sub.2), 3.97 (3H, s, OCH.sub.3),
3.91-3.3.87 (1H, m, CH), 3.82 (3H, s, OCH.sub.3), 2.92 (2H, bs,
OH).
4. Carbamate-Linked Nitrogen and Aniline Mustard Prodrugs
TABLE-US-00004 [0267] VG042-04 ##STR00062## VG0445-04
##STR00063##
benzofuran-2-ylmethyl 4-(bis(2-chloroethyl)amino)phenylcarbamate
(VG042-04)
##STR00064##
[0268] 2,2'-(4-nitrophenylazanediyl)diethanol (67)
[0269] Diethanolamine (2.70 mL, 2.5 mmol) was added to
1-fluoro-4-nitrobenzene (1.0 g, 7.09 mmol) in DMF (30 mL). The
resulting mixture was stirred at 140.degree. C. for 3.5 h. The
solution was cooled to room temperature and solvent was evaporated
off in vacuo. The residue was dissolved in EtOAc (30 mL) and washed
with water (3.times.10 mL) and brine (3.times.20 mL) and dried
(MgSO.sub.4). Solvent was evaporated off in vacuo and the product
was purified by flash chromatography, eluting with EtOAc to give 67
(400 mg, 25%) as a yellow solid. H.sup.1 NMR (500 MHz, CDCl.sub.3):
=8.06 (2H, d, J=9.50 Hz, ArH), 6.87 (2H, d, J=9.50 Hz, ArH), 4.27
(2H, t, J=5.35 Hz, 2.times.OH), 3.83 (4H, q, J=5.55 & 5.45 Hz,
2.times.CH.sub.2), 3.74 (4H, t, J=5.62 Hz, 2.times.CH.sub.2).
N,N-bis(2-(tert-butyldimethylsilyloxy)ethyl)-4-nitroaniline
(68)
[0270] To a cooled solution of 67 (400 mg, 1.77 mmol) and imidazole
(481 mg, 7.08 mmol) in DMF (10 mL) was added dropwise tert-butyl
dimethyl silyl chloride (2.72 mg, 3.54 mmol). The mixture was
allowed to reach room temperature and stirred for 48 h. The solvent
was evaporated off in vacuo and the product was purified by flash
chromatography, eluting with 10% EtOAc in ether to give 68 (200 mg,
25%) as a yellow solid. H.sup.1 NMR (500 MHz, CDCl.sub.3):
.delta.=8.06 (2H, d, J=9.45 Hz, ArH), 6.65 (2H, d, J=9.45 Hz, ArH),
3.80 (4H, t, J=5.80 Hz, 2.times.CH.sub.2), 3.62 (4H, t, J=5.80 Hz,
2.times.CH.sub.2), 0.85 (18H, s, 6.times.CH.sub.3), -0.01 (12H, s,
4.times.CH.sub.3).
benzofuran-2-ylmethyl 4-(bis(2-chloroethyl)amino)phenylcarbamate
(69) VG042-04
[0271] Compound 68 (200 mg, 0.44 mmol) was treated with hydrogen in
the presence of 10% Pd on carbon (20 mg). After 16 h stirring, the
mixture was filtered through Celite and the solvent was evaporated
off in vacuo to give the intermediate amino aniline product. This
was then reacted with triphosgene (195 mg, 0.70 mmol) in the
presence of triethylamine (260 uL, 0.70 mmol) in THF (15 mL). After
1 h stirring at room temperature a white precipitate was filtered
off and the solvent was evaporated off in vacuo to give a crude
residue of isocyanate aniline. This was used straight away in the
following step.
[0272] Isocyanate intermediate was dissolved in THF (10 mL). The
solution was cooled to 0.degree. C. Benzofuran-2-ylmethanol 59 (100
mg, 1.35 mmol) was added and the resulting mixture was stirred at
room temperature for 16 h. The mixture was cooled on ice and TBAF
(996 .mu.L, 3.38 mmol) was added dropwise over 5 min. The resulting
mixture was allowed to warm to room temperature and then stirred
for 20 min. THF was evaporated off in vacuo. The intermediate was
dissolved in pyridine (5 mL) and to this was added methane
sulphonyl chloride (12.5 .mu.L, 0.16 mmol). The mixture was stirred
at room temperature for 1 h. Pyridine was evaporated off in vacuo
and the crude product was purified by flash chromatography, eluting
with hexane:EtOAc (3:1) to give 69 (5 mg, 2%) as a white solid.
m/z=408.07 (M+H), 837.16 (2M+H).
benzofuran-2-ylmethyl bis(2-chloroethyl)carbamate (70) VG045-04
##STR00065##
[0274] A solution of 60 (200 mg, 0.64 mmol) in pyridine (3 mL) was
added to a solution of bis(2-chloroethylamine).hydrochloride (227
mg, 1.28 mmol) in pyridine (25 mL). The mixture was stirred at room
temperature for 16 h. DCM (10 mL) was added and the mixture was
washed with 2% citric acid solution (2.times.50 mL), water (50 mL),
brine (50 mL) and dried (MgSO.sub.4). Solvent was evaporated off in
vacuo and the product was purified by flash chromatography, eluting
with CH.sub.2Cl.sub.2:hexane (2:1) to give 70 (125 mg, 62%) as an
oil. m/z=338.05 (M+Na). H.sup.1 NMR (500 MHz, CDCl.sub.3):
.delta.=7.60 (1H, d, J=7.70 Hz, ArH), 7.51 (1H, d, J=8.05 Hz, ArH),
7.33 (1H, t, J=6.80 Hz, ArH), 7.26 (1H, t, J=6.80 Hz, ArH), 6.79
(1H, s, ArH), 5.28 (2H, s, CH.sub.2), 3.72-3.63 (8H, m,
4.times.CH.sub.2).
5. Ether-Linked Topoisomerase I Inhibitor Prodrug
##STR00066##
[0275] 5,7-Dimethoxybenzofuran-2-yl)methyl-camptothecin (71)
SU037-04
##STR00067##
[0277] Compound 41 (100 mg, 0.48 mmol) was dissolved in toluene (5
mL) and the solution was cooled to 0.degree. C. PBr.sub.3 (46
.mu.L, 0.48 mmol) was added dropwise over 10 min. The reaction
mixture was then brought up to room temperature and stirred for 1
h. The solvent was evaporated off in-vacuo. The crude residue was
used in the next step.
[0278] Sodium ethoxide (15 mg, 0.22 mmol) was added to DMF (5 mL)
at 0.degree. C., and the suspension was stirred for 10 min.
Camptothecin (81 mg, 0.22 mmol) was slowly added and resulting
mixture was stirred at this temp for 0.5 h. To this mixture, crude
residue from previous step, 2-(bromomethyl)-5,7-dimethoxybenzofuran
(50 mg, 0.18 mmol) was added portionwise. Resulting mixture was
stirred at room temperature for 2 h. DMF was evaporated off
in-vacuo and the residue was purified by flash chromatography,
eluting with DCM:EtOAc (2:1) to give the target compound as a white
solid (10 mg, 10%). m/z=555.19 (M+H).
6. Ether-Linked Tyrosine Kinase Inhibitor Prodrugs
TABLE-US-00005 [0279] VG048- 04 ##STR00068## SU01- A- 04
##STR00069## SU01-B- 04 ##STR00070## SU01- C-04 ##STR00071##
N-(4-(benzofuran-2-ylmethoxy)quinazolin-2-yl)-4,6,7-trimethylquinazolin-2--
amine (72) VG048-04
##STR00072##
[0281] Sodium ethoxide (3 mg, 0.05 mmol) was added to DMF (2 mL) at
0.degree. C., and the suspension was stirred for 5 min.
2-(4,6-dimethylquinazolin-2-ylamino)quinazolin-4-ol (15 mg, 0.05
mmol) was slowly added and resulting mixture was stirred at this
temperature for 0.5 h. To this mixture 2-(bromomethyl)benzofuran
(16 mg, 0.08 mmol) was added. Resulting mixture was stirred at room
temperature for 1 h. DMF was evaporated off in-vacuo to give a
crude white solid. This was purified by washing with cold ether and
EtOAc to give 72 (3 mg, 11%) as a white solid. m/z=462.2 (M+H).
7-(benzofuran-2-ylmethoxy)-5-isopropyl-2-methyl-[1,2,4]triazolo[1,5-a]pyri-
midine (73) SU01-A-04
##STR00073##
[0283] Sodium ethoxide (7.2 mg, 0.10 mmol) was added to DMF (2 mL)
at 0.degree. C., and the suspension was stirred for 5 min.
5-isopropyl-2-methyl-[1,2,4]triazolo[1,5-a]pyrimidin-7-ol (20 mg,
0.10 mmol) was slowly added and resulting mixture was stirred at
this temperature for 0.5 h. To this mixture
2-(bromomethyl)benzofuran (16 mg, 0.08 mmol) was added. Resulting
mixture was stirred at room temperature for 1 h. DMF was evaporated
off in-vacuo to give a crude white solid. This was purified by
semi-preparative HPLC to give 73 (6.3 mg, 19%) as a white solid.
m/z=323.13 (M+H), 645.27 (2M+H).
7-(benzofuran-2-ylmethoxy)-2-methyl-5-((4-methylpyrimidin-2-ylthio)methyl)-
-[1,2,4]triazolo[1,5-a]pyrimidine (74) SU01-B-04
##STR00074##
[0285] Sodium ethoxide (4.7 mg, 0.07 mmol) was added to DMF (2 mL)
at 0.degree. C., and the suspension was stirred for 5 min.
2-methyl-5-((4-methylpyrimidin-2-ylthio)methyl)-[1,2,4]triazolo[15-a]pyri-
midin-7-ol (20 mg, 0.07 mmol) was slowly added and resulting
mixture was stirred at this temp for 0.5 h. To this mixture 10 (16
mg, 0.08 mmol) was added. Resulting mixture was stirred at room
temperature for 1 h. DMF was evaporated off in-vacuo to give a
crude white solid. This was purified by semi-preprative HPLC to
give 74 (5.2 mg, 18%) as a white solid. m/z=419.09 (M+H), 837.23
(2M+H).
7-(benzofuran-2-ylmethoxy)-1-(2-fluorobenzyl)-4-methyl-1H-[1,2,3]triazolo[-
4,5-d]pyridazine (75) SU01-C-04
##STR00075##
[0287] Sodium ethoxide (5.2 mg, 0.08 mmol) was added to DMF (2 mL)
at 0.degree. C., and the suspension was stirred for 5 min
1-(2-fluorobenzyl)-4-methyl-1H-[1,2,3]triazolo[4,5-d]pyridazin-7-ol
(20 mg, 0.08 mmol) was slowly added and resulting mixture was
stirred at this temp for 0.5 h. To this mixture
2-(bromomethyl)benzofuran (16 mg, 0.08 mmol) was added. Resulting
mixture was stirred at room temperature for 1 h. DMF was evaporated
off in-vacuo to give a crude white solid. This was purified by
semi-preprative HPLC to give 75 (3 mg, 10%)as a white solid.
m/z=390.07 (M+H), 801.12 (2M+Na).
7. Carbamate-Linked Model Coumarin Prodrugs
TABLE-US-00006 [0288] TLE- M1- SU001C (.dagger.) ##STR00076##
SU030- 7-03 ##STR00077## SU002102 (.dagger.) ##STR00078## SU030-
8-03 (.dagger.) ##STR00079## VG032- 03 ##STR00080## SU033- 03
##STR00081## VG037- 03 ##STR00082## SU018- 03 ##STR00083## SU024-
2-03 ##STR00084## VG032- 05 ##STR00085## SU024- 3-03 (.dagger.)
##STR00086## VG036- 05 ##STR00087## SU030- 4-03 ##STR00088## VG041-
05 ##STR00089##
7-Isocyanato-4-methylcoumarin (76)
##STR00090##
[0290] A 200-mL three-neck flask fitted with a dry ice condenser
and magnetic stirrer was charged with a solution of 20% phosgene in
toluene solution (2.0 mL) and dioxane (80 mL). To this mixture was
added 7-amino-4-methyl-2H-chromen-2-one (2.00 g, 11.4 mmol). The
mixture was stirred at 100.degree. C. for 12 h. The initial yellow
colour disappeared and a white solid precipitated. An additional
20% phosgene in toluene solution (7.0 mL) was added and the mixture
heated for an additional 5 h, at which time the solution cleared.
Excess phosgene and traces of HCl was removed by bubbling nitrogen
gas through the solution. The cloudy solution was filtered to
remove unreacted 7-amino-4-methyl-2H-chromen-2-one and concentrated
to give 76 (0.5 g, 25%) as a white solid. H.sup.1 NMR (500 MHz,
CDCl.sub.3): .delta.=7.50 (1H, d, J=7.40 Hz, ArH), 7.46 (2H, s,
ArH), 6.20 (1H, s, ArH), 2.35 (3H, s, CH.sub.3): ir
(CH.sub.2CL.sub.2) 2314 (N.dbd.C.dbd.O), 1726 and 1615
cm.sup.-1.
naphthalen-1-ylmethyl 4-methyl-2-oxo-2H-chromen-7-ylcarbamate (77)
VG020-02
##STR00091##
[0292] Naphthalen-1-ylmethanol (56 mg, 0.28 mmol) and 76 (200 mg,
1.27 mmol) were dissolved in THF (2 mL). The resulting mixture was
stirred at room temperature for 15 min and then at 80.degree. C.
for 1 h. THF was evaporated off in vacuo. The residue was adsorbed
on silica and purified by flash chromatography, eluting with
CH.sub.2Cl.sub.2/hexane/EtOAc (1:1:1) to give 77 (3 mg, 5%) as a
white solid. m/z=360.14 (M+H), 719.27 (2M+H).
(2-chloroquinolin-3-yl)methyl
4-methyl-2-oxo-2H-chromen-7-ylcarbamate (78) SU030-7-03
##STR00092##
[0294] (2-chloroquinolin-3-yl)methanol (100 mg, 0.52 mmol) and 76
(155 mg, 0.77 mmol) were dissolved in THF (2 mL). The resulting
mixture was stirred at room temperature for 15 min and then at
80.degree. C. for 1 h. THF was evaporated off in-vacuo. The residue
was adsorbed on silica and purified by flash chromatography,
eluting with CH.sub.2Cl.sub.2/EtOAc (1:1) to give 78 (38 mg, 19%)
as a white solid. m/z=395.08 (M+H).
benzhydryl 4-methyl-2-oxo-2H-chromen-7-ylcarbamate (79)
SU0021-02
##STR00093##
[0296] (2-Chloroquinolin-3-yl)methanol (119 mg, 0.65 mmol) and 76
(70 mg, 0.35 mmol) were dissolved in THF (2 mL). The resulting
mixture was stirred at room temperature for 15 min and then at
80.degree. C. for 1 h. THF was evaporated off in vacuo. The residue
was adsorbed on silica and purified by flash chromatography,
eluting with CH.sub.2Cl.sub.2/EtOAc (1:1) to give 79 (50 mg, 37%)
as a white solid. m/z=386.16 (M+H).
benzhydryl 4-methyl-2-oxo-2H-chromen-7-ylcarbamate (80)
SU0021-02
##STR00094##
[0298] (4-Methyl-2-phenylpyrimidin-5-yl)methanol (100 mg, 0.50
mmol) and 76 (151 mg, 0.75 mmol) were dissolved in THF (2 mL). The
resulting mixture was stirred at room temperature for 15 min and
then at 80.degree. C. for 1 h. THF was evaporated off in vacuo. The
residue was adsorbed on silica and purified by flash
chromatography, eluting with CH.sub.2Cl.sub.2/EtOAc (1:1) to give
80 (70 mg, 35%) as a white solid. m/z=402.15 (M+H).
(1H-benzo[d]imidazol-2-yl)methyl
4-methyl-2-oxo-2H-chromen-7-ylcarbamate (81) VG032-03
##STR00095##
[0300] (1H-benzo[d]imidazol-2-yl)methanol (200 mg, 1.35 mmol) and
76 (272 mg, 1.35 mmol) were dissolved in THF (2 mL). The resulting
mixture was stirred at room temperature for 15 min and then at
80.degree. C. for 1 h. THF was evaporated off in vacuo. The residue
was adsorbed on silica and purified by flash chromatography,
eluting with CH.sub.2Cl.sub.2/EtOAc (1:1) to give 81 (80 mg, 17%)
as a white solid. m/z=350.12 (M+H).
(2H-chromen-3-yl)methyl 4-methyl-2-oxo-2H-chromen-7-ylcarbamate
(82) SU033-03
##STR00096##
[0302] 2H-chromene-3-carbaldehyde (500 mg, 3.13 mmol) was dissolved
in EtOH (10 mL). NaBH.sub.4 (119 mg, 3.13 mmol) was added
portionwise at 0.degree. C., with vigorous stirring. The suspension
was stirred at 0.degree. C. for 15 min and then at room temperature
for 1.5 h. Solvent was evaporated off in-vacuo to obtain the
alcohol intermediate as an oil. This was dissolved in THF (5 mL)
and 76 (155 mg, 0.77 mmol) was added. The resulting mixture was
stirred at room temperature for 15 min and then at 80.degree. C.
for 1 h. THF was evaporated off in vacuo. The residue was adsorbed
on silica and purified by flash chromatography, eluting with
CH.sub.2Cl.sub.2/EtOAc (1:1) to give 82 (80 mg, 8%) as a white
solid. m/z=364.12 (M+H), 727.23 (2M+H).
naphthalen-2-ylmethyl 4-methyl-2-oxo-2H-chromen-7-ylcarbamate (83)
VG037-03
##STR00097##
[0304] Naphthalen-2-ylmethanol (200 mg, 1.27 mmol) and 76 (279 mg,
1.39 mmol) were dissolved in THF (2 mL). The resulting mixture was
stirred at room temperature for 15 min and then at 80.degree. C.
for 1 h. THF was evaporated off in vacuo. The residue was adsorbed
on silica and purified by flash chromatography, eluting with
CH.sub.2Cl.sub.2/EtOAc (1:1) to give 83 (26 mg, 6%) as a white
solid. m/z=360.13 (M+H), 719.25 (2M+H).
benzofuran-2-ylmethyl 4-methyl-2-oxo-2H-chromen-7-ylcarbamate (84)
SU08-03
##STR00098##
[0306] Benzofuran-2-ylmethanol (300 mg, 2.03 mmol) and 76 (407 mg,
2.03 mmol) were dissolved in THF (2 mL). The resulting mixture was
stirred at room temperature for 15 min and then at 80.degree. C.
for 1 h. THF was evaporated off in vacuo. The residue was adsorbed
on silica and purified by flash chromatography, eluting with
CH.sub.2Cl.sub.2/EtOAc (1:1) to give 84 (130 mg, 18%) as a white
solid. m/z=350.09 (M+H), 699.17 (2M+H).
benzo[d]thiazol-2-ylmethyl 4-methyl-2-oxo-2H-chromen-7-ylcarbamate
(85) SU024-3-03
##STR00099##
[0308] Benzo[d]thiazol-2-ylmethanol (200 mg, 1.21 mmol) and 76 (365
mg, 1.8 mmol) were dissolved in THF (2 mL). The resulting mixture
was stirred at room temperature for 15 min and then at 80.degree.
C. for 1 h. THF was evaporated off in vacuo. The residue was
adsorbed on silica and purified by flash chromatography, eluting
with CH.sub.2Cl.sub.2/EtOAc (1:1) to give 85 (90 mg, 20%) as a
white solid. m/z=367.02 (M+H), 733.13 (2M+H).
4-(furan-2-yl)benzyl 4-methyl-2-oxo-2H-chromen-7-ylcarbamate (86)
SU024-3-03
##STR00100##
[0310] (4-(Furan-2-yl)phenyl)methanol (100 mg, 0.57 mmol) and 76
(139 mg, 0.69 mmol) were dissolved in THF (10 mL). The resulting
mixture was stirred at room temperature for 15 min and then at
80.degree. C. for 1 h. THF was evaporated off in vacuo. The residue
was adsorbed on silica and purified by flash chromatography,
eluting with CH.sub.2Cl.sub.2/EtOAc (1:1) to give the target
compound (20 mg, 9%) as a white solid. m/z=376.11 (M+H), 751.22
(2M+H).
(5-methylbenzo[b]thiophen-2-yl)methyl
4-methyl-2-oxo-2H-chromen-7-ylcarbamate (87) SU030-4-03
##STR00101##
[0312] (5-Methylbenzo[b]thiophen-2-yl)methanol (100 mg, 0.56 mmol)
and 76 (136 mg, 0.67 mmol) were dissolved in THF (2 mL). The
resulting mixture was stirred at room temperature for 15 min and
then at 50.degree. C. for 3 h. THF was evaporated off in vacuo. The
residue was adsorbed on silica and purified by flash
chromatography, eluting with CH.sub.2Cl.sub.2/EtOAc (1:1) to give
87 (38 mg, 18%) as a white solid. m/z=380.09 (M+H), 759.17
(2M+H).
(5-methoxybenzofuran-2-yl)methyl
4-methyl-2-oxo-2H-chromen-7-ylcarbamate (88) VG032-05
##STR00102##
[0314] (5-Methoxybenzofuran-2-yl)methanol 25 (200 mg, 1.12 mmol)
and 76 (190 mg, 0.95 mmol) were dissolved in THF (10 mL). The
resulting mixture was stirred at room temperature for 15 min and
then at room temperature for 16 h. THF was evaporated off in vacuo.
The residue was adsorbed on silica and purified by flash
chromatography, eluting with hexane/EtOAc (1:1) to give 88 (20 mg,
5%) as a white solid. m/z=380.13 (M+H), 759.26 (2M+H).
(5-bromobenzofuran-2-yl)methyl
4-methyl-2-oxo-2H-chromen-7-ylcarbamate (89) VG036-05
##STR00103##
[0316] (5-Bromobenzofuran-2-yl)methanol (100 mg, 0.44 mmol) and 76
(106 mg, 0.52 mmol) were dissolved in THF (2 mL). The resulting
mixture was stirred at room temperature for 15 min and then at
80.degree. C. for 1 h. THF was evaporated off in vacuo. The residue
was adsorbed on silica and purified by flash chromatography,
eluting with CH.sub.2Cl.sub.2/EtOAc (1:1) to give 89 (5.0 mg, 3%)
as a white solid. m/z=429.10 (M+H).
(5,7-dimethoxybenzofuran-2-yl)methyl
4-methyl-2-oxo-2H-chromen-7-ylcarbamate (90) VG041-05
##STR00104##
[0318] (5,7-dimethoxybenzofuran-2-yl)methanol 5 (50 mg, 0.24 mmol)
and 7-isocyanato-4-methylcoumarin (58 mg, 0.29 mmol) were dissolved
in THF (10 mL). The resulting mixture was stirred at room
temperature for 16 h. THE was evaporated off in vacuo. The residue
was adsorbed on silica and purified by flash chromatography,
eluting with CH.sub.2Cl.sub.2/EtOAc (1:1) to give 90 (5.0 mg, 3%)
as a white solid. m/z=410.04 (M+H).
8. Extended Linkers: Oxybenzyl Ether, Carbamate Benzyl Ether,
Oxybenzyl Carbamate
##STR00105## ##STR00106##
[0319]
4-methyl-7-(4-naphthalen-1-ylmethoxy)benzyloxy)-2H-chromen-2-one
(91) TLE-M1-SU001B
##STR00107##
[0321] A suspension of sodium ethoxide (924 mg, 13.6 mmol) in DMF
was stirred at 0.degree. C. for 10 min. Ethyl 4-hydroxy benzoate
(2.26 g, 13.6 mmol) was slowly added and resulting mixture was
stirred at this temp for 0.5 h, then allowed to reach room temp. To
this mixture 1-(bromomethyl)naphthalene (2.0 g, 9.0 mmol) was added
dropwise [(predissolved in DMF (5 mL)]. Resulting mixture was
stirred at room temperature for 16 h. DMF was evaporated off
in-vacuo and the residue taken up in EtOAc, and washed with brine,
water and 1M NaOH (2.times.30 mL). The organic layer was dried
(MgSO.sub.4) and the solvent evaporated off in-vacuo to yield ethyl
4-(naphthalene-1-ylmethoxy)benzoate (1.7 g, 5.55 mmol). This was
then dissolved in THF and LiAlH.sub.4 (211 mg, 5.55 mmol) was added
portionwise, with vigorous stirring. The suspension was stirred at
room temperature for 3 h. THF was evaporated off in-vacuo. The
crude residue was taken up in EtOAc and washed with water, brine
and dried (MgSO.sub.4). EtOAc was evaporated off in-vacuo to obtain
(4-(naphthalene-1-ylmethoxy)phenyl)methanol (1.3 g, 4.9 mmol) as a
crude product. This was used in the next reaction step without
further purification.
[0322] (4-(naphthalen-1-ylmethoxy)phenyl)methanol (1.0 g, 3.8 mmol)
was dissolved in toluene (30 mL) and pyridine (305 uL, 3.8 mmol)
was added. The solution was cooled to 0.degree. C. PBr.sub.3 (359
uL, 3.8 mmol) was added dropwise over 15 min. The mixture was
brought up to room temperature and stirred for 1 h. It was washed
with K.sub.2CO.sub.3 solution and extracted with EtOAc (3.times.30
mL). The EtOAc layer was washed with brine and dried (MgSO.sub.4).
The solvent was evaporated off in-vacuo to obtain
1-((4-(bromomethyl)phenoxy)methyl)naphthalene (660 mg, 53%). This
intermediate was used in the following reaction.
[0323] Sodium ethoxide (156 mg, 2.29 mmol) was added to DMF at
0.degree. C., and the suspension was stirred for 10 min.
7-Hydroxy-4-methylcoumarin (403 mg, 2.29 mmol) was slowly added and
resulting mixture was stirred for 0.5 h, and then allowed to reach
room temperature. To this mixture
1-((4-(bromomethyl)phenoxy)methyl)naphthalene (500 mg, 1.53 mmol)
was added portionwise. Resulting mixture was stirred at room
temperature for 16 h. DMF was evaporated off in-vacuo and the
residue was taken up in EtOAc, and washed with brine (2.times.50
mL), water (2.times.50 mL) and 1M NaOH (2.times.30 mL). The organic
layer was dried (MgSO.sub.4) and purified by flash chromatography,
eluting with hexane: EtOAc (2:1) to give 91 (200 mg, 31%) as a
white solid, Mpt=154-156.degree. C.; H.sup.1 NMR (500 MHz,
acetone-d.sub.6): .delta.=8.10 (1H, d, ArH), 8.00-7.99 (2H, m, Ar),
7.97-7.71 (2H, m, ArH), 7.70-7.53 (3H, m, ArH), 7.24 (1H, s, ArH),
7.09 (1H, d, CH), 6.23 (1H, s, CH), 5.68 (2H, s, CH.sub.2), 2.40
(3H, s, CH.sub.3). .sup.13C NMR (500 MHz, acetone-d.sub.6,
DEPT135): .delta.=154.6 (qC), 153.4 (qC), 133.3 (qC), 131.1, 129.8,
128.9, 128.7, 128.5, 126.9, 126.7, 126.5, 126.4, 126.0, 125.9
(11.times.Ar CH), 125.3, (Ar CH), 123.8 (Ar CH), 114.8 (Ar CH),
113.1 (Ar CH), 112.7 (Ar CH), 111.2 (Ar CH), 111.1 (Ar CH), 101.7
(CH), 69.6 & 67.9 (2.times.CH.sub.2), 18.1 (CH.sub.3).
7-(4-(benzhydryloxy)benzyloxy)-4-methyl-2H-chromen-2-one (92)
TLE-M1-SU004B
##STR00108##
[0325] Sodium ethoxide (661 mg, 9.7 mmol) was added to DMF (5 mL)
at 0.degree. C. Resulting suspension was stirred for 15 min. Ethyl
4-hydroxy benzoate (1.61 g, 9.7 mmol) was slowly added and
resulting mixture was stirred at this temperature for 0.5 h, then
allowed to reach room temperature. To this mixture diphenylmethyl
bromide (2.0 g, 8.1 mmol) was added portionwise. Resulting reaction
mixture was stirred at room temperature for 16 h. DMF was
evaporated off in-vacuo and the residue was taken up in EtOAc, and
washed with brine, water and 1M NaOH (2.times.30 mL). The organic
layer was dried (MgSO.sub.4) and the solvent evaporated off
in-vacuo to yield ethyl 4-(benzhydryloxy)benzoate (1.0 g, 3.0
mmol). This was then dissolved in THF (5 mL) and LiAlH.sub.4 (114
mg, 3.0 mmol) was added portionwise, with vigorous stirring. The
suspension was stirred at room temperature for 3 h. THE was
evaporated off in-vacuo. The crude residue was taken up in EtOAc
and washed with water, brine and dried (MgSO.sub.4). EtOAc was
evaporated off in-vacuo to obtain (4-(benzhydryloxy)phenyl)methanol
(760 mg, 2.62 mmol) as a crude product. This was used in the next
reaction step without further purification.
[0326] (4-(Benzhydryloxy)phenyl)methanol (500 mg, 1.72 mmol) was
dissolved in toluene (20 ml) and pyridine (139 uL, 1.72 mmol) was
added. The solution was cooled to 0.degree. C. PBr.sub.3 (163 uL,
1.72 mmol) was added dropwise over 15 min. The resulting mixture
was stirred at room temperature for 1 h. It was then washed with
saturated K.sub.2CO.sub.3 solution and extracted with EtOAc
(3.times.30 mL). The EtOAc layer was washed with brine and dried
(MgSO.sub.4). The solvent was evaporated off in-vacuo to obtain
((4-(bromomethyl)phenoxy)methylene)dibenzene as an oil, (450 mg,
74%). This intermediate was used in the following reaction.
[0327] Sodium ethoxide (87 mg, 1.28 mmol) was added to DMF (3 mL)
at 0.degree. C., and the suspension was stirred for 10 min.
7-Hydroxy-4-methylcoumarin (225 mg, 1.28 mmol) was slowly added and
resulting mixture was stirred at this temperature for 0.5 h, then
allowed to reach room temperature. To this mixture
((4-(bromomethyl)-phenoxy) methylene)dibenzene (300 mg, 1.53 mmol)
was added portionwise. Resulting mixture was stirred at room
temperature for 16 h. DMF was evaporated off in-vacuo and the
residue was taken up in EtOAc, and washed with brine (2.times.50
mL), water (2.times.50 mL) and 1M NaOH (2.times.30 mL). The organic
layer was dried (MgSO.sub.4) and purified by flash chromatography,
eluting with hexane: EtOAc (2:1) to give 92 (80 mg, 21%) as a white
solid. Mpt=179-181.degree. C. H.sup.1 NMR (500 MHz,
acetone-d.sub.6): .delta.=7.66 (1H, d, ArH), 7.56 (4H, d, ArH),
7.39-7.34 (6H, m, ArH), 7.08 (2H, d, ArH), 6.97 (2H, d, ArH), 6.93
(2H, d, ArH), 6.51 (1H, s, CH), 6.12 (1H, s, CH), 5.12 (2H, s,
CH.sub.2), 2.42 (3H, s, CH.sub.3).
7-(4-(benzofuran-2-ylmethoxy)benzyloxy)-4-methyl-2H-chromen-2-one
(94) SU010B-02
##STR00109##
[0328] Ethyl 4-(benzofuran-2-ylmethoxy)benzoate (93)
[0329] Sodium ethoxide (580 mg, 8.5 mmol) was added to DMF (10 mL)
at 0.degree. C. Resulting suspension was stirred for 15 min. Ethyl
4-hydroxy benzoate (1.4 g, 8.5 mmol) was slowly added and resulting
mixture was stirred at this temperature for 0.5 h, then allowed to
reach room temp. To this mixture 10 (1.5 g, 7.1 mmol) was added
dropwise, predissolved in DMF (5 mL). Resulting reaction mixture
was stirred at room temperature for 2 h. DMF was evaporated off
in-vacuo and the residue was taken up in EtOAc, and washed with
brine, water and 1M NaOH (2.times.30 mL). The organic layer was
dried (MgSO.sub.4) and the solvent evaporated off in-vacuo to give
93 as a white solid (920 mg, 44%). m/z=423.18 (M+H). H.sup.1 NMR
(500 MHz, Acetone-d.sub.6): .delta.=8.0 (2H, d, ArH), 7.60 (1H, d,
ArH), 7.30-7.15 (2H, m, ArH), 7.27-7.19 (1H, m, CH), 7.00 (2H, d,
ArH), 6.80 (1H, s, CH), 5.20 (2H, s, CH.sub.2), 4.10 (2H, q,
CH.sub.2), 1.25 (3H, t, CH.sub.3).
7-(4-(benzofuran-2-ylmethoxy)benzyloxy)-4-methyl-2H-chromen-2-one
(94) U010B-02
[0330] Compound 93 (400 mg, 1.29 mmol) was dissolved in THF (15 mL)
and LiAlH.sub.4 (49 mg, 1.29 mmol) was added portionwise, with
vigorous stirring. The suspension was stirred at room temperature
for 1 h. THE was evaporated off in-vacuo. The crude residue was
taken up in EtOAc and washed with water, brine and dried
(MgSO.sub.4). EtOAc was evaporated off in-vacuo to obtain
(4-(benzofuran-2-ylmethoxy)phenyl)methanol (220 mg, 67%) as a crude
product. This was dissolved in toluene (10 mL). The solution was
cooled to 0.degree. C. PBr.sub.3 (98 .mu.L, 1.04 mmol) was added
dropwise over 15 min. The resulting mixture was stirred at room
temperature for 1 h. It was then washed with saturated
K.sub.2CO.sub.3 solution and extracted with EtOAc (3.times.30 mL).
The EtOAc layer was washed with brine and dried (MgSO.sub.4). The
solvent was evaporated off in-vacuo to obtain
2-((4-(bromomethyl)phenoxy)methyl)benzofuran as a colourless oil,
(132 mg). This intermediate was used in the following reaction.
[0331] Sodium ethoxide (43 mg, 0.63 mmol) was added to DMF at
0.degree. C., and the suspension was stirred for 10 min.
7-Hydroxy-4-methylcoumarin (110 mg, 0.63 mmol) was slowly added and
resulting mixture was stirred at this temperature for 0.5 h, then
allowed to reach room temperature. To this mixture 93 (132 mg, 0.42
mmol) was added portionwise. Resulting reaction mixture was stirred
at room temp for 16 h. DMF was evaporated off in-vacuo and the
residue was taken up in EtOAc, and washed with brine (2.times.50
mL), water (2.times.50 mL) and 1M NaOH (2.times.30 mL). The organic
layer was dried (MgSO.sub.4) and purified by flash chromatography,
eluting with hexane: EtOAc (2:1) to give 94 (80 mg, 47%) as a white
solid. Mpt=153-155.degree. C. m/z=413 (M+H). H.sup.1 NMR (500 MHz,
Acetone-d.sub.6): .delta.=7.56-7.46 (2H, m, CH), 7.40 (1H, t,
J=8.2, CH), 7.34 (2H, d, J=8.50, CH), 7.27-7.19 (1H, m, CH),
7.14-7.09 (1H, m, CH), 7.00-6.98 (2H, d, CH, J=11.8), 6.86-6.80
(3H, m, CH), 5.99 (1H, s, CH), 5.14 (2H, s, CH.sub.2), 5.03 (2H, s,
CH.sub.2), 2.28 (3H, s, CH.sub.3).
naphthalen-1-ylmethy 4-((4-methy-2-oxo-2H-chromen-7
yloxy)methyl)phenylcarbamate (95) VG021-03
##STR00110##
[0333] To a stirred solution of naphthalene methanol (4.0 g, 25.3
mmol) in THF (30 mL) was added TEA (100 uL). To this was added
dropwise, ethylcyanobenzoate (4.0 g, 21.0 mmol), predissolved in
THF (10 mL). The resulting solution was stirred at room temperature
for 16 h. Solvent was evaporated off to give a crude intermediate,
ethyl 4-((naphthalen-1-ylmethoxy)carbonylamino)benzoate (1.3 g).
This was then dissolved in THF (15 mL) and LiAlH.sub.4 (141 mg,
3.75 mmol) was added portionwise, with vigorous stirring. The
suspension was stirred at room temperature for 1 h. THF was
evaporated off in-vacuo. The crude residue was taken up in EtOAc
and washed with water, brine and dried (MgSO.sub.4). EtOAc was
evaporated off in-vacuo to obtain (naphthalen-1-ylmethyl
4-(hydroxymethyl)phenylcarbamate (500 mg, 1.62 mmol), as a crude
product. This was dissolved in toluene (10 mL). The solution was
cooled to 0.degree. C. PBr.sub.3 (154 uL, 1.62 mmol) was added
dropwise over 15 min. The resulting mixture was stirred at room
temperature for 1 h. The solvent was evaporated off in-vacuo to
obtain naphthalen-1-ylmethyl 4-(bromomethyl)phenylcarbamate as an
oil, (300 mg). This intermediate was used in the following reaction
without further purification.
[0334] Sodium ethoxide (44 mg, 0.65 mmol) was added to DMF at
0.degree. C., and the suspension was stirred for 10 min.
7-Hydroxy-4-methylcoumarin (114 mg, 0.70 mmol) was slowly added and
the mixture was stirred at this temperature for 0.5 h, then allowed
to reach room temperature. To this mixture
2-((4-(bromomethyl)phenoxy)methyl)benzofuran (200 mg, 0.42 mmol)
was added portionwise. Resulting mixture was stirred at room
temperature for 16 h. DMF was evaporated off in-vacuo and the
residue was taken up in EtOAc, and washed with brine (2.times.50
mL), water (2.times.50 mL) and 1M NaOH (2.times.30 mL). The organic
layer was dried (MgSO.sub.4) and purified by flash chromatography,
eluting with hexane: EtOAc (2:1) to give 95 (160 mg, 63%) as a
white solid; Mpt=153-155.degree. C. m/z=488.2 (M+H). H.sup.1 NMR
(500 MHz, Acetone-d.sub.6): .delta.=7.56-7.46 (2H, m, CH), 7.40
(1H, t, J=8.2, CH), 7.34 (2H, d, J=8.50, CH), 7.27-7.19 (1H, m,
CH), 7.14-7.09 (1H, m, CH), 7.00-6.98 (2H, d, CH, J=11.8),
6.86-6.80 (3H, m, CH), 5.99 (1H, s, CH), 5.14 (2H, s, CH.sub.2),
5.03 (2H, s, CH.sub.2), 2.28 (3H, s, CH.sub.3).
4-(benzofuran-2-ylmethoxy)benzyl
4-methyl-2-oxo-2H-chromen-7-ylcarbamate (96) SU024-1-03)
##STR00111##
[0336] Compound 93 (300 mg, 1.01 mmol) was dissolved in THF (15 mL)
and LiAlH.sub.4 (38 mg, 1.01 mmol) was added portionwise, with
vigorous stirring. The suspension was stirred at room temperature
for 1 h. THF was evaporated off in-vacuo. The crude residue was
taken up in EtOAc and washed with water, brine and dried
(MgSO.sub.4). EtOAc was evaporated off in-vacuo to obtain
(4-(benzofuran-2-ylmethoxy)phenyl)methanol (150 mg, 0.59 mmol), as
a crude product. This was used in the next step without further
purification.
[0337] Sodium ethoxide (40 mg, 0.59 mmol) was added to DMF at
0.degree. C., and the suspension was stirred for 10 min.
(4-(benzofuran-2-ylmethoxy)phenyl)methanol (150 mg, 0.59 mmol), was
added and resulting mixture was stirred at this temperature for 0.5
h, then allowed to reach room temperature. To this mixture 76 (130
mg, 0.65 mmol) was added portionwise. Resulting mixture was stirred
at room temperature for 2 h. DMF was evaporated off in-vacuo. The
crude residue was purified by flash chromatography, eluting with
hexane: EtOAc (2:1) to give the target compound (20 mg, 8%) as a
white solid. m/z=456.10 (M+H). H.sup.1 NMR (500 MHz, DMSO-d.sub.6):
.delta.=10.22 (1H, bs, NH), 7.69-7.64 (2H, m, ArH), 7.58 (1H, d,
J=8.15 Hz, ArH), 7.55 (1H, s, ArH), 7.42 (2H, d, J=8.00 Hz, ArH),
7.33 (1H, t, J=7.70 Hz, ArH), 7.26 (1H, t, J=7.5 Hz, ArH), 7.11
(2H, d, J=8.28 Hz, ArH), 7.06 (1H, s, ArH), 6.23 (1H, s, ArH), 5.29
(2H, s, CH.sub.2), 5.12 (2H, s, CH.sub.2), 2.37 (3H, s,
CH.sub.3).
7-(4-((5-methoxybenzofuran-2-yl)methoxy)benzyloxy)-4-methyl-2H-chromen-2-o-
ne (97) VG040-05
##STR00112##
[0339] Sodium ethoxide (62 mg, 0.90 mmol) was added to DMF (3 mL)
at 0.degree. C. Resulting suspension was stirred for 15 min. Ethyl
4-hydroxy benzoate (148 mg, 0.90 mmol) was slowly added and
resulting mixture was stirred at this temperature for 0.5 h, then
allowed to reach room temperature. To this mixture 26 (180 mg, 0.75
mmol) was added. Resulting reaction mixture was stirred at room
temperature for 1 h. DMF was evaporated off in-vacuo to give a
crude intermediate (150 mg). This was then dissolved in THF (5 mL)
and LiAlH.sub.4 (34 mg, 0.90 mmol) was added portionwise, with
vigorous stirring. The suspension was stirred at room temperature
for 1 h. THF was evaporated off in-vacuo. The crude residue was
taken up in EtOAc and washed with water, brine and dried
(MgSO.sub.4). EtOAc was evaporated off in-vacuo to obtain
(4-((5-methoxybenzofuran-2-yl)methoxy)phenyl)methanol (120 mg) as a
crude product. This was dissolved in toluene (4 mL). The solution
was cooled to 0.degree. C. PBr.sub.3 (84 .mu.L, 1.04 mmol) was
added dropwise over 5 min. The resulting mixture was stirred at
room temperature for 1 h. The solvent was evaporated off in-vacuo
to obtain 2-((4-(bromomethyl)phenoxy)methyl)-5-methoxybenzofuran as
a colourless oil, (90 mg). This intermediate was used in the
following reaction.
[0340] Sodium ethoxide (22 mg, 0.31 mmol) was added to DMF (2 mL)
at 0.degree. C., and the suspension was stirred for 10 min.
7-Hydroxy-4-methylcoumarin (54 mg, 0.31 mmol) was slowly added and
resulting mixture was stirred at this temperature for 0.5 h. To
this mixture 2-((4-(bromomethyl)phenoxy)methyl)-5-methoxybenzofuran
(90 mg, 0.23 mmol) was added portionwise. Resulting reaction
mixture was stirred at room temp for 16 h. DMF was evaporated off
in-vacuo and the residue was purified by flash chromatography,
eluting with hexane: EtOAc (1:1) to give 94 (22 mg, 7%) as a white
solid. m/z=443 (M+H).
4-(benzhydryloxy)benzyl 4-methyl-2-oxo-2H-chromen-7-ylcarbamate
(98) SU032-02
##STR00113##
[0342] Sodium ethoxide (300 mg, 4.05 mmol) was added to DMF (10 mL)
at 0.degree. C. Resulting suspension was stirred for 10 min. Ethyl
4-hydroxy benzoate (739 mg, 4.05 mmol) was slowly added and
resulting mixture was stirred at this temperature for 20 min. To
this mixture diphenylmethyl bromide (1.0 g, 4.05 mmol) was added
portionwise. Resulting reaction mixture was stirred at room
temperature for 16 h. DMF was evaporated off in-vacuo and the
residue was taken up in EtOAc, and washed with water and brine. The
organic layer was dried (MgSO.sub.4) and the solvent evaporated off
in-vacuo to yield ethyl 4-(benzhydryloxy)benzoate (830 mg). This
was then dissolved in THF (5 mL) and LiAlH.sub.4 (114 mg, 3.0 mmol)
was added portionwise, with vigorous stirring. The suspension was
stirred at room temperature for 3 h. THF was evaporated off
in-vacuo. The crude residue was taken up in EtOAc and washed with
water, brine and dried (MgSO.sub.4). EtOAc was evaporated off
in-vacuo to obtain (4-(benzhydryloxy)phenyl)methanol (760 mg, 2.62
mmol) as a crude product. This was used in the next reaction step
without further purification.
[0343] A solution of (4-(Benzhydryloxy)phenyl)methanol (100 mg,
0.34 mmol) and 76 in toluene (10 ml). was refluxed for 2 h. The
reaction was allowed to cool to room temperature and the resulting
precipitate formed was filtered and washed with cold ether and
EtOAc to give 98 (100 mg, 60%) as a white solid. m/z=491.55
(M+H).
Biological Activity
Example 1: CYP1B1 Metabolism of Prodrugs
Substituent Effect on the Fragmentation of Benzofuran Ether and
Carbamate Linked Coumarins by CYP1 Isoenzymes and Human Liver
Microsomes (HLM).
[0344] Commercially available Supersomal.TM. CYP1A1, CYP1A2,
CYP1B1, and pooled human liver microsomes (supplied BD Gentest,
Oxford, UK) comprised an enzymatic screen to identify structure
activity relationships (SARs) underlying the structural features
which control the efficiency and selectivity of prodrug
fragmentation by CYP1B1 expressed in cancer relative to cytochrome
P450 enzymes expressed in normal tissues including the liver. HLMs
are derived from human patient liver and according to the supplier
contain a battery of cytochrome P450s including CYP1A2, CYP2A6,
CYP2B6, CYP2B6, CYP2C8, CYP2C9, CYP2D6, CYP2E1, CYP3A4, and CYP4A
but not CYP1A1 or CYP1B1.
[0345] Typical Supersomal.TM. CYP1A1, CYP1A2, CYP1B1 enzyme
metabolism studies used 10 pmol enzyme, 100 .mu.mol dm.sup.-3
NADPH, in 10 mmol dm.sup.-3 potassium phosphate buffer at pH 7.4
and 37.degree. C. Supersomal.TM. enzyme metabolism was started by
adding a stock solution of prodrug dissolved in DMSO to give a
final concentration of 10 .mu.mol dm.sup.-3 prodrug and 0.5% DMSO.
HLM screening used 60 microlitre microsomes, 100 .mu.mol dm.sup.-3
NADPH, in 10 mmol dm.sup.-3 potassium phosphate buffer at pH 7.4
and 37.degree. C., in 1.5 ml total reaction volume.
[0346] Compounds of the invention comprise a series of
heteroaromatic triggers coupled to ether and carbamate linkers to
the hydroxyl group of 7-hydroxy-4-methycoumarin and
7-amino-4-methylcoumarin, respectively. Further examples of the
invention comprise compounds were heteroaromatic triggers are
coupled via the so-called extended oxybenzyl ether linker
(--Ar--CH(Z.sup.7)X.sup.3--=-phenyl-CH.sub.2O--) to the hydroxyl
group of 7-hydroxy-4-methycoumarin. Further examples of the
invention comprise compounds where heteroaromatic triggers are
coupled via the so-called extended oxybenzyl carbamate linker to
the amino group of 7-amino-4-methycoumarin. Further examples of the
invention comprise compounds where heteroaromatic triggers are
coupled via a carbamate benzyl ether linker to the hydroxyl group
of 7-hydroxy-4-methycoumarin.
[0347] Both 7-hydroxy-4-methycoumarin and 7-amino-4-methylcoumarin
are partially deprotonated at physiological pH 7.4 and both
coumarin anions are highly fluorescent with fluorescence emission
wavelength maxima of 450 and 445 nm, respectively. When the
coumarins are coupled to hetero-aromatic triggers via linkers
described in this invention the fluorescence of the coumarin anion
is quenched. Therefore, enzymatic hydroxylation of the
hetero-aromatic trigger and resultant linker fragmentation can be
monitored in real time by release of the coumarin anion by kinetic
fluorimetry. This prodrug design strategy has been successfully
used to monitor the fragmentation of so-called bioreductive
hypoxia-activated prodrugs by P450 reductase not to be confused
with CYP1B1 which is a mono-oxygenase enzyme (See, e.g.: Everett S
A et al., Modifying rates of reductive elimination of leaving
groups from indolequinone prodrugs: a key factor in controlling
hypoxia-selective drug release", Biochem Pharmacol., 63: 1629-39,
2002).
[0348] Release of the coumarin anion indicative of linker
fragmentation was monitored using a 1 cm path length fluorescence
cell in a Cary Eclipse kinetic flourimeter with excitation and
emission slits set at 5 nm. Coumarin anion release from compounds
of the invention was detected at the excitation wavelength
.lamda..sub.ex=350-nm and the emission wavelength
.lamda..sub.em=450 nm. Change in fluorescence intensity was
quantified against a linear calibration plot of fluorescence
intensity versus coumarin concentration (0 to 3.5 .mu.mol
dm.sup.-3) in 10 mmol dm.sup.-3 potassium phosphate buffer at pH
7.4 using the same instrument settings as for enzyme
metabolism.
[0349] Specific fragmentation activities (in pmol coumarin
min.sup.-1 pmol cytochrome P450.sup.-1) for the CYP1 isoenzyme and
HLM-activated fragmentation and release of coumarin from benzofuran
ether and carbamate-linked coumarins are shown in Table 3. An
electron donating substituent (Me, MeO) or electron withdrawing
substituents (Cl, Br, F) in one or both of the 5- and 7-position of
the benzofuran has a significant effect on both fragmentation
specificity and efficiency. The 4- and 6-positions on the
benzofuran are left unsubstituted (R.sup.4 and R.sup.6.dbd.H) as
they are likely positions for enzymatic hydroxylation necessary to
induce ether or carbamate linker fragmentation according to the
proposed mechanism. The structure activity relationship (SAR)
governing the substituent effect at the 5- and 7-position on the
benzofuran and CYP1 isoenzyme-induced fragmentation efficiency and
selectivity are not predictable for either ether or carbamate
linker fragmentation.
[0350] SU10A (see Table 3 below) where Z.sup.3.dbd.H, Z.sup.5.dbd.H
bearing an ether-linked coumarin is fragmented by CYP1A1, CYP1A2,
and CYP1B1 as well as HLM. For HLM the inclusion of 10 .mu.mol
dm.sup.-3 .alpha.-naphthoflavone (a CYP1-selective enzyme
inhibitor) inhibits SU10A fragmentation indicating that CYP1A2 is
solely responsible for HLM-mediated coumarin release. Benzofuran is
therefore a generic trigger moiety that can facilitate the
fragmentation of ether-linked prodrugs by CYP1 isoenzymes.
[0351] Electron withdrawing substituents on the benzofuran in
VG016-04 (see also Table 3 below) where Z.sup.3.dbd.F,
Z.sup.5.dbd.F inhibits CYP1 isoenzyme and HLM-induced fragmentation
of the ether linker. However, electron donating substituents in
VG035-05 (see also Table 3 below) where Z.sup.3=MeO, Z.sup.5=MeO
results in CYP1B1-specific fragmentation of the ether bond as no
linker fragmentation is observed for CYP1A or HLM. The specific
fragmentation activity for VG035-05 with CYP1B1 is 13.65.+-.1.00
pmol coumarin min.sup.-1 pmol cytochrome P450.sup.-1 is the highest
efficiency of the various benzofuran ether-linked coumarins
investigated (see Table 3 below). The 5,7-dimethoxybenzofuran
moiety can therefore be used to specifically trigger the
fragmentation of ether-linked prodrugs by CYP1B1 an enzyme which is
over-expressed in cancer.
[0352] In marked contrast to VG035-04 (which contains an
ether-linked coumarin), VG041-05 (which contains the corresponding
carbamate-linked coumarin) where Z.sup.3=MeO, Z.sup.5=MeO is
selectively fragmented by CYP1A1 (but not CYP1A2 or CYP1B1) with a
specific fragmentation activity=5.51.+-.0.06 pmol coumarin
min.sup.-1 pmol cytochrome P450.sup.-1. According to Table 3 below
all compound examples of structure B containing a carbamate linker
are fragmented by CYP1A1 but not CYP1B1. The only exception is
VG032-05 where X.sup.3=MeO, Z.sup.5=MeO giving a CYP1B1 specific
fragmentation activity=1.53.+-.0.09 pmol coumarin min.sup.-1 pmol
cytochrome P450.sup.-1, which is .about.6-fold lower than VG027-05
which contains an ether linker.
Example 2: Combining the Model Prodrug Library with a CYP1B1
Substrate Prediction Model Links Substrate Specificity to Prodrug
Activation and Fragmentation
Performing a High-Throughput Screen (HTS) to Build a Bioactivity
Dataset for CYP181
[0353] The target enzyme CYP1B1 was screened against two commercial
libraries including the ChemDiv Diversity 50,000 test compound
collection and the ChemDiv Kinase Targeted 10,000 test compound
collection with a view to identifying activity differentiating
substructures and a large bioactivity dataset from which to build a
substrate specificity model. The HTS was performed in miniaturized
384-well format using a liquid handler (Beckman FXp), bulk
dispensers (Matrix Wellmate) and a luminescent plate reader
(Molecular Devices Analyst AD plate Reader). P450-Glo.TM. Assays
provide a luminescent method for measuring cytochrome P450
activity. A conventional reaction is performed by incubating the
human supersomal CYP1B1 plus reductase (BD Gentest.TM., UK)
recombinant enzyme with a luminogenic cytochrome P450 substrate,
namely Luciferin 6' chloroethyl ether (Luciferin-CEE) which is a
substrate for CYP181 but not for luciferase. Luciferin-CEE is
converted to a luciferin product that is detected in a second
reaction with Luciferin Detection Reagent (CYP1B1 Luminescent Assay
Kit, P450-Glo.TM. from Promega, Madison, USA). The reagent
simultaneously stops the cytochrome P450 reaction and initiates a
stable luminsescent signal with a half-life >2 h. The amount of
light produced in the second reaction is proportional to the
activity of CYP1B1. The biochemical end-point was substrate
inhibition of the enzyme (0.5 pmol/well) working at the apparent
K.sub.m for Luciferin-CEE (20 .mu.mol dm.sup.-3). The assay is
characterized by excellent Z'-factors typically greater than 0.6
(where a Z'=1.0 denotes a perfectly robust highly reproducible
assay) when run in 384-well format. The negative control was the
level of activity which defined the unmodified state of the enzyme
target while the positive control was the level of activity which
defined a hit. The negative control contained the
CYP1B1/KPO.sub.4/NADPH/substrate reaction mixture and an equivalent
concentration of 1% DMSO used for solubilization of the test
compounds. The positive control in the assay contained the
CYP1B1/KPO.sub.4/NADPH substrate reaction mixture with
.alpha.-naphthoflavone which completely inhibits CYP1B1 enzyme
activity at a final concentration of 5 .mu.mol dm.sup.-3. The
positive and negative controls were deposited in the outer columns
of every 384-well plate with the test compounds deposited in the
remaining 320 wells. The definition of a hit is a test compound
that is a substrate inhibitor CYP1B1 activity by 80-100% at a
concentration of 0.5 .mu.mol dm.sup.-3.
[0354] Pipeline Pilot (Scitegic, San Diego, USA) was used to
streamline and integrate the large quantity of data to identify
SARs from the CYP1B1 HTS, supported by computational scientists at
the UCSF SMDC. The software was used to identify (1) preliminary
SAR results of hits versus non-hits, (2) determine physicochemical
properties e.g. molecular weight, calculated log P, H-atom
donor/acceptor interactions of the hit population, (3) determine
the frequency of ring fragments and functional groups, and (4)
define an in silico model for the prediction of CYP1B1 substrate
inhibition as a basis for future prodrug design. Importantly, s
significant number of the hits.about.10% had a molecular weight
between 400-500, the latter being the maximum molecular weight of
test compounds available in both compound collections. This
information defined the maximum molecular weight of the prodrug
permissible whilst maintaining CYP1B1 substrate specificity.
Structural analysis of hit scaffolds in SARvision v2 from
CHEMAPPS.TM. (La Jolla, Calif., USA) confirmed that test compounds
did not support the correct functional group (e.g. a trigger
hydroxymethyl substituent) for direct integration into the coupling
chemistry reviewed in scheme 1. However, by identifying ring
fragments of high frequency in hits versus non-hits it was possible
to identify templates for trigger moieties which could then be
functionalized appropriately for coupling reactions.
An in Silico Model for Predicting Cytochrome P450 Substrate
Inhibition in Support of Prodrug Design.
[0355] A major challenge in prodrug design is to define the
strategy to integrate the trigger, linker and effector chemistry
whilst maintaining substrate specificity for the target enzyme. The
CYP1B1 HTS was extremely valuable in identifying potential trigger
moieties but subsequent `hit to lead` chemistry incorporating a
linker and effector drug could mean that the final prodrug
structure would not be optimal for target enzyme activation.
Optimal usage of the vast amount of structural data from the two
HTS screens (totaling 60,000 test compounds) was achieved by
developing an in silico prediction model of cytochrome P450 1B1
substrate inhibition using Gaussian Kernel weighted k-nearest
neighbour (k-NN) algorithm based on Tanimoto similarity searches on
extended connectivity fingerprints. The optimal parameters of the
CYP1B1 kernel weighted k-NN model were chosen using leave-one-out
cross validation on a training set selected from 45,000 and 9,000
test compounds from the ChemDiv Diverse and Kinase libraries. The
remainder of the test compounds, 6,000 in total, were used as an
internal test set to confirm the accuracy of the model to predict
substrate inhibition. Any test compounds exhibiting >20 but
<80% inhibition were designated non-classified. The model
accurately predicted 89% of the classified non-substrate inhibitors
and 95% of the classified substrate inhibitors. CYP1B1 substrate
prediction model protocol was uploaded into the Scitegic Web Port
to facilitate the docking of putative prodrug structures through an
interface to standard chemistry drawing packages such as
ChemDraw/IsisDraw.
Validation of the CYP1B1 Substrate Prediction Model Using an
External Test Set of Compounds
[0356] A 384-well stock plate constituting an external test set for
the CYP1B1 substrate prediction model was constructed and included:
(1) known CYP1B1 substrate inhibitors including, for example,
tetramethoxystilbene, .beta.-estradiol, .alpha.-napthoflavone,
ethoxyresorufin, resveratrol, (2) compounds which are not CYP1B1
substrate inhibitors including, for example, quinidine (a potent
specific inhibitor of CYP2D6), sulfaphenazole (a potent specific
inhibitor of CYP2C9), and (3) the model prodrugs VG016-05 and
VG035-05, and (4) and the phosphoramidate mustard prodrugs SU025-04
and SU046-04. The external test set stock concentration was 10 mmol
dm.sup.-3 in DMSO and the percentage CYP1B1 substrate inhibition at
a final concentration of 0.5 mmol dm.sup.-3 was determined using
the same methods described for the main CYP1B1 HTS. Experiments
were perfomed in triplicate to give a mean % substrate inhibition
of CYP1B1 activity.+-.standard deviation. All the external test set
structures were submitted as queries to the CYP1B1 substrate
prediction model via the Scitegic Web Port to generate predicted
values of % substrate inhibition of CYP1B1 in order to compare with
the actual biochemical measurement of % substrate inhibition.
[0357] The comparative actual and predicted % substrate inhibition
for CYP1B1 values were as follows:
TABLE-US-00007 % CYP1B1 substrate inhibition compound actual
predicted accuracy tetramethoxystilbene 95.76 .+-. 0.33 97.34 98%
.beta.-estradiol, 72.34 .+-. 0.45 76.12 95% .alpha.-napthoflavone
99.12 .+-. 0.23 92.45 93% ethoxyresorufin 92.13 .+-. 0.56 87.23 95%
resveratrol 72.34 .+-. 0.56 76.45 95% sulfaphenazole 2.89 .+-. 0.15
4.25 68% quinidine 1.63 .+-. 0.34 2.56 64% VG016-05 2.45 .+-. 0.32
2.98 82% VG035-05 97.34 .+-. 0.32 93.56 96% SU025-04 91.22 .+-.
0.48 87.36 96% SU046-04 96.45 .+-. 0.22 92.34 96%
[0358] The CYP1B1 substrate prediction model was accurate in
predicting % substrate inhibition of CYP1B1 across multiple classes
of compound with a broad range of activity confirming validation of
the model using an external test set of compounds. Importantly, in
terms of an inventive step the model was accurately able to predict
the activity of the two model prodrugs VG016-05 and VG035-05 in
terms of CYP1B1 substrate inhibition which can be linked directly
to the efficiency of prodrug fragmentation and release of the
7-hydroxy-4-methy coumarin anion. According to Table 3 electron
donating or electron withdrawing substituents in the R.sup.5 and
R.sup.7 of the benzofuran trigger activate these model prodrugs to
aromatic hydroxylation and fragmentation. When R.sup.5 and
R.sup.7.dbd.F, i.e. electron withdrawing substituents as in
VG016-05 the model prodrug is not activated by CYP1B1 as accurately
predicted and as a consequence there is no fragmentation of the
linker. In marked contrast when R.sup.5 and R.sup.7=MeO, i.e.
electron donating substituents as in VG035-05 the model prodrug is
activated by CYP1B1 as accurately predicted resulting in
fragmentation of the linker with high efficiency. Incorporation of
the dimethoxybenzofuran trigger moiety into the phosphoramidate
mustard prodrugs SU025-04 and SU046-04 generate compound which are
accurately predicted to be good substrate inhibitors of CYP111. In
conclusion, the combination of model prodrug libraries and CYP1B1
substrate prediction models based on a database of CYP1B1
bioactivity facilitate the design of specific CYP1B1-activated
prodrugs.
Example 3: Prodrug Cytotoxicity in Wild-Type CHO Cells and CHO
Cells Engineered to Express CYP1A1 and CYP1B1 Isozymes
[0359] Engineered CHO cells were used to demonstrate selective cell
killing mediated by CYP1 expression. In the experiments described
below, compounds were exposed to wild-type CHO cells engineered to
express either CYP1A1 (CHO/CYP1A1) or CYP1B1 (CHO/CYP1B1)
enzymes.
[0360] CHO cells: Chinese Hamster Ovary (CHO) DUKXB11 cells were
grown under standard cell culture conditions in .alpha.-MEM
supplemented with 10% FCS, 1 unit/ml each of hypoxanthine and
thymidine, and penicillin (100 IU/ml) and streptomycin (100
.mu.g/ml) according to literature methods (Ding S, et al., Arch.
Biochem. Biophys., 348: 403-410, 1997, the contents of which are
incorporated herein by reference). Cells were grown at 37.degree.
C. in a humidified atmosphere plus 5% CO.sub.2.
[0361] CHO/CYP1A1 and CHO/CYP1B1 cells: CHO cells containing
recombinant CYP1A1 and recombinant CYP1B1 co-expressing P450
reductase, namely (CHO/CYP1A1) and (CHO/CYP1B1) respectively, were
cultured using the standard culture medium for CHO cells
supplemented with 0.4 mg/ml G418 disulfate salt and 0.3 .mu.M
methotrexate (Sigma/Aldrich Co., Gillingham, Dorset, UK) according
to methods described in the literature (ibid.) Cells were grown at
37.degree. C. in a humidified atmosphere plus 5% CO.sub.2.
Recombinant CYP1A1 and CYP1B1 Expression
[0362] Dihydrofolate reductase (DHFR) gene amplification of either
human cDNA CYP1A1 or cDNA CYP1B1 in CHO cells was used to achieve
high levels of functional enzyme when co-expressed with human P450
reductase (ibid.; Ding S, et al., Biochem J., 356(Pt 2): 613-9,
2001). Modified CYP1A1 or CYP1B1 cDNA was digested and ligated into
to the mammalian expression vector pDHFR to generate the plasmids
pDHFR/1A1 and pDHFR/1B1, respectively (ibid.) Cell culture and DNA
transfection into CHO DUKXB11 was carried out according to methods
described in the literature and transfected cells selected for the
DHFR+ phenotype by growth in nucleoside deficient medium (ibid.)
DHFR+ clones were pooled, and grown on increasing concentrations of
MTX (0.02 to 0.1 .mu.M) for amplification of transfected CYP1A1 or
CYP1B1 cDNA. Cell clones that survived 0.1 mM MTX selection were
isolated then further selected with 0.3 .mu.M MTX. The resulting
cell lines were analysed for CYP1A1 or CYP1B1 expression by
immunoblotting. Cell lines expressing a high level of each enzyme
were stably transfected with plasmid pcDNA/HR containing a full
length human cytochrome P450 reductase (CPR) cDNA, and selected
with G418 (0.8 mg/ml) and MTX (0.3 .mu.M) according to methods
described in the literature (ibid.) After isolation of resistant
clones the concentration of G418 was reduced to 0.4 mg/ml and the
homogeneity of the cell lines assured by repeated cloning. The CHO
cell line transfected with the plasmid carrying cDNA CYP1A1
subsequently transfected with CPR cDNA was designated CHO/CYP1A1
and the CHO cell line transfected with the plasmid carrying cDNA
CYP1B1 subsequently transfected with CPR cDNA was designated
CHO/CYP1B1.
Immunochemical Detection of CYP1A1 and CYP1B1
[0363] Cells were harvested and lysed by sonication using standard
methods in the literature (Ding S, et al., 1997, the contents of
which are incorporated herein by reference). Proteins (typically 50
.mu.g of lysate) were separated by SDS/PAGE, transferred to a
nitrocellulose membrane and probed using standard methods (Paine M
J, et al., Arch. Biochem. Biophys., 328: 380-388, 1996, the
contents of which are also incorporated herein by reference). Human
CYP1A1 plus reductase Supersomes.TM., human CYP1A2 plus reductase
Supersomes.TM. and CYP1B1 plus reductase Supersomes.TM. (BD
Biosciences, Oxford, UK) were used as positive controls (typically
0.03 to 0.3 pmole) for immunochemical detection of enzyme
expression in cell lines. A WB-1B1 primary antibody (dilution
1:1500, BD Biosciences, Oxford, UK) and an anti-CYP1A2 antibody
which cross reacts with CYP1A1 (dilution 1:2000, Cancer Research
Technology, London, UK) were used to detect CYP1B1 and CYP1A1
expression, respectively. The secondary antibody was goat
anti-rabbit IgG used at a 1:500 dilution. Immunoblots were
developed using the Enhanced Chemiluminescence (ECL) Western-blot
detection kit (GE Healthcare Life Sciences, Amersham,
Buckinghamshire, UK).
Western-Blot Characterization of CYP1A1 and CYP1B1 Expression in
Engineered CHO Cells
[0364] FIG. 1a of the accompanying drawings is a typical
western-blot showing the detection of CYP1B1 protein expression in
lysate from the CHO/CYP1B1 cell line which is detectable in neither
the untransfected CHO DUKXB11 cells nor the CHO/CYP1A1 cell line.
The band corresponds to a molecular weight of 56 kDa and matches
the band for human CYP1B1 Supersomal.TM. enzyme. FIG. 1b is a
typical western-blot showing the detection of CYP1A1 protein
expression in lysate from the CHO/CYP1A1 cell line which is
detectable in neither the untransfected CHO DUKXB11 cells nor the
CHO/CYP1B1 cell line. The band corresponds to a molecular weight of
60 kDa and matches the band for human CYP1A1 Supersomal.TM. enzyme
detected by the cross reactivity of the anti-CYP1A2 antibody.
Functional CYP1 Enzyme Activity
[0365] The ethoxyresorufin O-deethylation (EROD) assay is widely
used to confirm functional CYP1 activity (Chang T K and Waxman D J,
"Enzymatic Analysis of cDNA-Expressed Human CYP1A1, CYP1A2, and
CYP1B1 with 7-Ethoxyresorufin as Substrate", Methods Mol. Biol.,
320: 85-90, 2006, the contents of which are incorporated herein by
reference). The assay determines O-dealkylation of
7-ethoxyresorufin by CYP1A1, CYP1A2, and CYP1B1 to generate the
enzymatic product resorufin, which is monitored continuously by
fluorescence emission at 580 nm. An alternative assay for measuring
enzyme activity is the commercially available Promega P450-Glo.TM.
Assay utilizing Luciferin-CEE as a luminogenic substrate for CYP1
enzymes in Cali J J, et al., Expert. Opin. Drug Metabolism
Toxicol., 2(4): 629-45, 2006, the contents of which are also
incorporated herein by reference. The EROD assay and Promega
P450-Glo.TM. Assay with selective and non-selective CYP1 inhibitors
were used to confirm that the CHO cell lines referred to above were
expressing the expected CYP1 enzymes in a functional form.
[0366] In the absence of inhibitors, CHO/CYP1A1 and CHO/CYP1B1 (but
not wild-type CHO cells) converted 7-ethoxyresorufin to resorufin
or Luciferin-CEE to luciferin, thereby confirming functional CYP1
expression in these cells (see Table 1 below).
[0367] As expected, addition of the broad-spectrum CYP1 inhibitor,
.alpha.-naphthoflavone, abolished activity in both CYP1 expressing
cell lines (see Table 1 below). The selective inhibitor,
tetramethoxystibene, is 30-fold selective for CYP1B1 over CYP1A1
(Chun Y J, Kim S, Kim D, Lee S K and Guengerich F P, "A New
Selective and Potent Inhibitor of Human Cytochrome P450 1B1 and its
Application to Antimutagenesis", Cancer Res 61(22): 8164-70, 2001).
Tetramethoxystilbene abolished activity at high concentrations in
both CYP1 expressing cell lines and preferentially decreased
activity in CYP1B1 expressing cells (compared with CYP1A1
expressing cells) cells at lower concentrations (see Table 1
below).
[0368] These results provide independent confirmation that the
CYP1A1 and CYP1B1 expression levels are as expected.
Determining Cytotoxicity IC.sub.50 Values in CHO, CHO/CYP1A1 and
CHO/CYP1B1 Cell Lines
[0369] A single cell suspension of CHO, CHO/CYP1A1 or CHO/CYP1B1 in
100 .mu.l of the required cell culture medium was seeded onto
96-well plates at a cell density of 1500 cells per well and placed
in an incubator for 24 h at 37.degree. C. The stock solution of
test compound in DMSO was then added to give a concentration range
of 100, 30, 10, 3, 1, 0.3, 0.1, 0.03, 0.01, 0.003, 0.001, 0 .mu.M.
The final concentration of DMSO 0.2% was found not to affect the
growth characteristics of the various CHO cell lines. The cells
were incubated with the test compound for 72 or 96 h after which
all the medium was aspirated and replaced with 100 .mu.l of fresh
medium to compensate for the loss of medium due to evaporation. The
cells were incubated with 20 .mu.l of the MTS assay reagent for 1.5
h and the absorbance per well at 510 nm measured using a plate
reader. The mean absorbance and standard deviation for each test
compound concentration was calculated versus a series of controls
including (a) cells plus medium, (b) cell plus medium containing
DMSO 0.2%, (c) medium alone, and (d) medium containing DMSO 0.2%
and a range of test compound concentrations from 0 to 100 .mu.mol
dm.sup.-3. The cytotoxicity IC.sub.50 value was calculated from the
plot of the percentage cell growth (where 100% cell growth
corresponds to untreated control cells) versus test compound
concentration.
[0370] Cytotoxicity IC.sub.50 values are defined herein as the
concentration of compound which kills 50% of cells and fold
selectivity is calculated by dividing the IC.sub.50 in non-CYP1
expressing cells with the IC.sub.50 in CYP1A1 or CYP1B1 expressing
cells. Differential cytotoxicity IC.sub.50 ratios are calculated
from compound IC.sub.50 in normal CHO cells divided by IC.sub.50 in
CYP1A1 or CYP1B1 transfected CHO cells.
Promega.TM. CellTiter 96.RTM. Aqueous Non-Radioactive Cell
Proliferation (MTS) Assay
[0371] The commercially available MTS assay is a homogeneous,
colorimetric method for determining the number of viable cells in
proliferation, cytotoxicity or chemosensitivity assays. The assay
is composed of solutions of tetrazolium compound
[3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl-
)-2H-tetrazolium, inner salt; MTS] and an electron coupling reagent
(phenazine methosulfate) PMS. MTS is bioreduced by cells into a
formazan product that is soluble in tissue culture medium. The
absorbance of the formazan product at 510 nm can be directly
measured from 96-well assay plates. The quantity of formazan
product as measured by the amount of absorbance at 490 or 510 nm is
directly proportional to the number of living cells in culture.
[0372] Two compounds of the invention (SU025-04 and SU046-04), are
designed to release the phosphoramidate mustards
N,N-bis(2-chloro-ethyl)phosphoramide (CI-IPM) and
N,N-bis(2-bromo-ethyl)phosphoramide (Br-IPM), respectively, when
activated by CYP1B1. The high toxicities of the two phosphoramidate
mustards CI-IPM and Br-IPM are significantly reduced when
incorporated in the prodrugs SU025-04 and SU046-04, respectively.
Both SU025-04 and SU046-04 have cytotoxicity IC.sub.50 values no
less than 10 .mu.mol dm.sup.-3 in wild-type CHO cells at 72 or 96 h
exposure in marked contrast to CI-IPM and Br-IPM which have
cytotoxicity IC.sub.50 values below 0.007 .mu.mol dm.sup.-3 in
wild-type CHO cells after a 72 h exposure (see Table 2 below). The
mechanism of activation of the two prodrugs can be deduced from
their comparative cytotoxicity IC.sub.50 values in CHO-wild-type
(which lacks CYP1 enzyme expression), CHO/1A1, and CHO/CYP1B1
cells. For example, SU025 and SU046 exhibit low toxicity in
wild-type CHO cells but are highly toxic to CHO/1B1 cells giving
differential cytotoxicity IC.sub.50 ratios of 1689 and 5075,
respectively at 72 h exposure. At a longer exposure time of 96 h
the CYP1B1-selective prodrugs SU025-04 and SU046-04 are 3367 and
5400-fold more toxic to CYP1B1 expressing cells than non-CYP1B1
expressing cells (see Table 2 below). Compounds SU025-04 and
SU046-04 are therefore demonstrably CYP1B1-activated prodrugs.
SU025-04 and SU046-04 exhibit similarly low cytotoxicity to
wild-type CHO and CHO/CYP1A1 cells with differential cytotoxicity
IC.sub.50 ratios <1 at 72 h exposure indicating that the highly
toxic phosphoramidate mustards are not released by CYP1A1
activation (see Table 2 below). As expected from the literature the
two clinically used prodrugs ifosfamide and cyclophosphamide which
also generate alkylating isophosphoamidate mustards when activated
by CYP2B6 and CYP3A4 but not CYP1 enzymes (e.g.: McFadyen M C,
Melvin W T and Murray G I, "Cytochrome P450 Enzymes: Novel Options
for Cancer Therapeutics", Mol Cancer Ther., 3(3): 363-71, 2004) are
both non-toxic at the highest concentration of 100 .mu.mol
dm.sup.-3 and longest 96 h exposure times used in this cytotoxicity
assay (see again Table 2 below).
Example 4: Prodrug Cytotoxicity in Primary Human Tumour Cell
Lines
[0373] Prodrug Cytotoxicity in a Primary Human Head and Neck
Squamous Cell Carcinoma Tumour Cell Line (UT-SCC-14) which
Constitutively Expresses CYP1B1
[0374] Greer, et al., in Proc. Am. Assoc. Cancer Res., 45: 3701,
2004, reported that CYP1B1 was over-expressed during the malignant
progression of head and neck squamous cell carcinoma (HNSCC) but
not in normal epithelium. A primary UT-SCC-14 tumour cell line was
isolated from a cancer patient with HNSCC (see e.g. Yaromina et.
al., Radiother Oncol., 83: 304-10, 2007 and Hessel et al., Int J
Radiat Biol., 80; 719-27, 2004. The patient was a male, aged 25,
with an HNSCC characterized by the following clinicopathological
parameters: location, scc linguae; T.sub.3 N.sub.1, M.sub.0; site,
tongue; lesion, primary; grade G2. The UT-SCC-14 cell line
constitutively expresses CYP1B1 at the mRNA and protein level and
was used to demonstrate compound cytotoxicity in cancer cell
derived from a human cancer characterised by over-expression of
CYP1B1 (Greer, et al., in Proc. Am. Assoc. Cancer Res., 45: 3701,
2004).
[0375] UT-SCC-14 tumour cells: The HNSCC cell line was grown under
standard cell culture conditions in EMEM (500 ml) supplemented with
foetal calf serum (50 ml), non-essential amino acids (100.times., 5
ml), sodium pyruvate (100 mmol dm.sup.-3, 5 ml), L-glutamine (200
mmol dm.sup.-3, 5 ml) with penicillin 100 IU/ml/streptomycin (100
.mu.g/ml, 5 ml) according to literature methods (Hessel et al., Int
J Radiat Biol., 80; 719-27, 2004, the contents of which are
incorporated herein by reference).
Determining Prodrug Cytotoxicity IC.sub.50 Values in Primary Nead
and Neck Tumour Cell Lines
[0376] A UT-SCC-14 tumour cell suspension at 2000 cells per well on
a 96-well plate and if necessary add fresh media to give a total
volume per well of 100 .mu.l. The cells were allowed to attach for
4 h in an incubator. After 4 h it was confirmed that the cells had
adhered to the bottom of the 96-well plate under a microscope, then
the medium was removed and replaced with fresh medium containing a
stock solution of the test compound in ethanol to give the
following final concentrations 0, 0.001, 0.003, 0.01, 0.03, 0.1,
0.3, 1, 3, 10, 30, 100 .mu.mol dm.sup.-3 at a final volume of 100
.mu.l per well. The final concentration of ethanol 0.2% was found
not to effect the growth characteristics of the UT-SCC-14 cell
line. The UT-SCC-14 cells were incubated with test compound for 72
h after which time all aspirated and replaced with 100 .mu.l of
fresh medium to compensate for the loss of medium due to
evaporation. The cells were incubated with 20 .mu.l of the MTS
assay reagent for 1.5 h and the absorbance per well at 510 nm
measured using a plate reader. The mean absorbance and standard
deviation for each test compound concentration was calculated
versus a series of controls including (a) cells plus medium, (b)
cell plus medium containing ethanol 0.2%, (c) medium alone, and (d)
medium containing ethanol 0.2% and a range of test compound
concentrations from 0 to 100 .mu.mol dm.sup.-3. The cytotoxicity
IC.sub.50 value was calculated from the plot of the percentage cell
growth (where 100% cell growth corresponds to untreated control
cells) versus test compound concentration.
[0377] Cytotoxicity IC.sub.50 values are defined herein as the
concentration of compound which kills 50% of the UT-SCC-14 tumour
cells. The commercially available MTS assay is a homogeneous,
colorimetric method for determining the number of viable cells in
proliferation, cytotoxicity or chemosensitivity assays and was used
as described previously in this Example 3 above.
[0378] Two compounds of the invention (SU025-04 and SU046-04), are
designed to release the phosphoramidate mustards
N,N-bis(2-chloro-ethyl)phosphoramide mustard (CI-IPM) and
N,N-bis(2-bromo-ethyl)phosphoramide mustard (Br-IPM), respectively,
when activated by CYP1B1. The cytotoxicity IC.sub.50 values for
SU025-04 and SU046-04 in the UT-SCC-14 tumour cells after 72 h
exposure were 0.05.+-.0.01 .mu.mol dm.sup.-3 and 0.02.+-.0.01
.mu.mol dm.sup.-3, respectively. The data show the potent
cytotoxicity of SU025-04 and SU046-04 in the UT-SCC14 cell line
from a cancer patient with HNSCC which over-expresses CYP1B1.
[0379] SU025-04 and SU046-04 were evaluated in 3 additional primary
head and neck cell lines including the UT-SCC-8, the UT-SCC-9 and
the UTSCC-10 cultured under the same conditions as the UT-SCC-14.
For SU025-04 the cytotoxicity IC.sub.50 in .mu.mol dm.sup.-3 were
UT-SCC-8 (0.31.+-.0.06), UT-SCC-9 (0.43.+-.0.07), UT-SCC-10
(0.22.+-.0.03) after a 72 h exposure. For SU025-04 the cytotoxicity
IC.sub.50 in .mu.mol dm.sup.-3 were UT-SCC-8 (0.06.+-.0.02),
UT-SCC-9 (0.15.+-.0.02), UT-SCC-10 (0.09.+-.0.03) after a 72 h
exposure. The data indicate that SU046-04 is a more potent
cytotoxin than SU025-04 across a range of primary head and neck
cell lines which contitutively express CYP1B1.
[0380] One compound of the invention, SU037-04, was designed to
release camptothecin when activated by CYP1B1. For SU037-04 the
cytotoxicity IC.sub.50 in mol dm.sup.-3 for each primary tumour
cell line was UT-SCC-8 (0.56.+-.0.04), UT-SCC-9 (0.22.+-.0.08),
UT-SCC-10 (0.21.+-.0.04), UT-SCC-14 (0.12.+-.0.07) after a 72 h
exposure.
[0381] One compound of the invention, SU048-04, was designed to
release gemcitabine when activated by CYP1B1. The cytotoxicity
IC.sub.50 for SU048-04 in the UT-SCC-14 tumour cell line was
0.94.+-.0.02 .mu.mol dm.sup.-3 after a 72 h exposure. Co-incubation
with .alpha.-napthoflavone (a potent CYP1B1 inhibitor) at 10
.mu.mol dm.sup.-3 significantly reduced the toxicity of SU048-04 to
12.2.+-.0.2 .mu.mol dm.sup.-3 thereby providing indirect evidence
for the activation of the prodrug by CYP1B1 constitutively
expressed in the cells.
Example 5: Anti-Tumour Activity of SU046-04 in a Primary Human
Tumour Xenograft Model which Constitutively Expresses CYP1B1
[0382] Primary UTSCC-14 cell lines 3.times.10.sup.6 were implanted
subcutaneously in the flank of nude mice. Mice were randomized to
10 animals per group when the tumour volume was 100 to 150
mm.sup.3. SU046-04 was given intraperitoneally at 12, 25 and 50
mg/Kg in PBS versus vehicle alone for 2 cycles: daily for 5 days/2
days off. Tumour volume was measured every 4 days using calipers. A
significant inhibition of tumour growth was observed in all three
treatment arms compared to the vehicle alone. Tumour growth delay
at 28 days was 31% at 12 mg/Kg, 56% at 25 mg/Kg, and 90% at 50
mg/Kg with 4/10 complete responses. No observed adverse effects or
significant body weight loss after highest exposure 250 mg/Kg.
TABLE-US-00008 TABLE 1 Specific CYP1 enzyme activity in Chinese
Hamster Ovary (CHO) cells stably transfected with CYP1A1 or CYP1B1
determined using both fluorogenic and luminogenic substrates
.sup.aSpecific activity/pmol resorufin or luciferin min.sup.-1 mg
protein.sup.-1 CHO CHO/CYP1A1 CHO/CYP1B1 ethoxyresorufin
Luciferin-CEE ethoxyresorufin Luciferin-CEE ethoxyresorufin
Luciferin-CEE nd nd 29 .+-. 4 21 .+-. 3 19 .+-. 3 22 .+-. 4
chemical inhibitor .sup.b.alpha.-naphthoflavone 10 .mu.mol
dm.sup.-3 nd nd nd nd nd nd .sup.cTMS 5 .mu.mol dm.sup.-3 nd nd nd
nd nd nd 10 nmol dm.sup.-3 nd nd 26 .+-. 4 20 .+-. 5 5 .+-. 2 3
.+-. 2 .sup.aMeasured by two methods including the fluoresecent
7-ethoxyresorufin O-deethylation (EROD) assay or the Promega
P450-Glo .TM. Assay utilizing Luciferin-CEE as a luminogenic
substrate for CYP1 enzymes. Luciferin-CEE for CYP1A1 K.sub.mapp ~30
.mu.mol dm.sup.-3 Luciferin-CEE for CYP1B1 K.sub.mapp ~20 .mu.mol
dm.sup.-3 Ethoxyresorufin for CYP1A1and CYP1B1 K.sub.m ~0.27
.mu.mol dm.sup.-3 nd = no detectable activity.
.sup.b.alpha.-naphthoflavone inhibits all CYP1 enzymes at 10
.mu.mol dm.sup.-3. .sup.cTetramethoxystilbene (TMS) is a selective
inhibitor of CYP1B1 with IC.sub.50 of 6 nmol dm.sup.-3 30-fold
greater than CYP1A1 and inhibits both enzymes at >1 .mu.mol
dm.sup.-3.
TABLE-US-00009 TABLE 2 In vitro cytotoxicity (IC.sub.50) of
prodrugs in a Chinese Hamster Ovary (CHO) cell line stably
transfected with CYP1A1 or CYP1B1 compared to isophosphoramide
mustard (IPM), cyclophosphamide, and ifosfamide.
.sup.a,b,cCytotoxicity (IC.sub.50)/.mu.mol dm.sup.-3 CHO CHO/CYP1B1
IC.sub.50 ratio CHO/CYP1A1 IC.sub.50 ratio CHO CHO/CYP1B1 IC.sub.50
ratio Compound 72 h 72 h 72 h 72 h 72 h 96 h 96 h 96 h SU025-04
15.2 .+-. 0.2 0.009 .+-. 0.005 1689 25.5 .+-. 1.0 <1 10.1 .+-.
0.3 0.003 .+-. 0.002 3367 CI-IPM 0.007 .+-. 0.004 0.008 .+-. 0.005
<1 0.006 .+-. 0.002 1.2 SU046-04 20.3 .+-. 1.2 0.004 .+-. 0.002
5075 20.3 .+-. 2.1 <1 16.2 .+-. 0.2 0.003 .+-. 0.001 5400 Br-IPM
0.005 .+-. 0.002 0.004 .+-. 0.002 1.25 0.005 .+-. 0.003 1
ifosfamide ND ND ND ND ND ND ND ND cyclophosphamide ND ND ND ND ND
ND ND ND .sup.aCytotoxicity measured using the Promega CellTiter 96
.RTM. AQ.sub.ueous Non-Radioactive Cell Proliferation Assay.
.sup.bExposure time was 72 or 96 h, dose range 0 to 100 .mu.mol
dm.sup.-3. .sup.cIC.sub.50 ratios calculated from compound
IC.sub.50 in normal CHO cells divided by IC.sub.50 in CYP1A1 or
CYP1B1 transfected CHO cells. ND = not detectable, indicating
<50% toxicity observed at highest compound concentration
tested.
TABLE-US-00010 TABLE 3 Substituent effect on the fragmentation of
benzofuran ether and carbamate linked coumarins by CYP1 enzymes and
human liver microsomes (HLM). ##STR00114## .sup.a,bSpecific
fragmentation activity/pmol coumarin m.sup.-1 pmol cytochrome
P450.sup.-1 Compound Structure Substituent CYP1A1 CYP1A2 CYP1B1 HLM
SU010A A R.sup.5 = H; R.sup.7 = H 20.91 .+-. 00.34 8.66 .+-. 0.28
5.19 .+-. 0.15 22.8 .+-. 0.69 VG015-05 A R.sup.5 = F; R.sup.7 = H
19.35 .+-. 3.08 nd 3.16 .+-. 0.28 nd VG016-05 A R.sup.5 = F;
R.sup.7 = F no release no release no release no release VG017-05 A
R.sup.5 = F; R.sup.7 = Me 5.39 .+-. 1.34 nd 2.95 .+-. 0.59 nd
VG027-05 A R.sup.5 = MeO; R.sup.7 = H 88.10 .+-. 5.69 20.93 .+-.
0.59 11.83 .+-. 0.15 8.98 .+-. 0.49 VG029-05 A R.sup.5 = H; R.sup.7
= MeO 19.53 .+-. 1.07 nd 6.45 .+-. 1.15 nd VG035-04 A R.sup.5 = Br;
R.sup.7 = H 28.08 .+-. 1.98 nd 8.85 .+-. 0.28 nd VG028-05 A R.sup.5
= Cl; R.sup.7 = H 17.52 .+-. 0.83 nd 6.67 .+-. 0.01 nd VG035-05 A
R.sup.5 = MeO; R.sup.7 = MeO no release no release 13.65 .+-. 1.00
no release SU018-03 B R.sup.5 = H; R.sup.7 = H 2.41 .+-. 0.18 no
release no release no release VG032-05 B R.sup.4 = MeO; R.sup.7 = H
12.19 .+-. 3.02 no release 1.53 .+-. 0.09 3.21 .+-. 0.25 VG036-05 B
R.sup.5 = Br; R.sup.7 = H no release no release no release no
release VG041-05 B R.sup.5 = MeO; R.sup.7 = MeO 5.51 .+-. 0.06 no
release no release no release .sup.aEther or carbamate linker
fragmentation was monitored by coumarin anion release by kinetic
fluorimetry using excitation/emission wavelengths: .lamda.ex = 350
nm/.lamda.em = 450 nm. CYP1 enzyme concentration was 10 pmol and
the volume of HLM was 60 .mu.l a final reaction volume of 1.5 ml.
Specific fragmentation activities are quoted as the mean .+-.
standard deviation of three measurements. nd = not determined.
* * * * *