U.S. patent application number 11/336623 was filed with the patent office on 2008-02-14 for pyrrolobenzodiazepines.
This patent application is currently assigned to GOVERNMENT OF THE U.S.A. REPRESENTED BY THE SECRETARY, DEPT. OF HEALTH AND HUMAN SERVICES. Invention is credited to Stephen Gregson, Philip Howard, Paul S. Liu, Kenneth M. Snader, David E. Thurston, B. Rao Vishnuvajjala.
Application Number | 20080039448 11/336623 |
Document ID | / |
Family ID | 34553801 |
Filed Date | 2008-02-14 |
United States Patent
Application |
20080039448 |
Kind Code |
A1 |
Liu; Paul S. ; et
al. |
February 14, 2008 |
Pyrrolobenzodiazepines
Abstract
A compound of formula I: ##STR1## or solvate thereof, wherein n
is 1 to 10, and M and M' are independently selected from monovalent
pharmaceutically acceptable cations, or together represent a
divalent pharmaceutically acceptable cation.
Inventors: |
Liu; Paul S.; (Chevy Chase,
MD) ; Vishnuvajjala; B. Rao; (Rockville, MD) ;
Snader; Kenneth M.; (Vero Beach, FL) ; Thurston;
David E.; (Hampshire, GB) ; Howard; Philip;
(St. Albans, GB) ; Gregson; Stephen; (London,
GB) |
Correspondence
Address: |
LEYDIG, VOIT & MAYER, LTD.
TWO PRUDENTIAL PLAZA, SUITE 4900
180 NORTH STETSON AVENUE
CHICAGO
IL
60601-6731
US
|
Assignee: |
GOVERNMENT OF THE U.S.A.
REPRESENTED BY THE SECRETARY, DEPT. OF HEALTH AND HUMAN
SERVICES
Rockville
MD
|
Family ID: |
34553801 |
Appl. No.: |
11/336623 |
Filed: |
January 20, 2006 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
|
|
11129207 |
May 13, 2005 |
7244724 |
|
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11336623 |
Jan 20, 2006 |
|
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PCT/GB04/04497 |
Oct 22, 2004 |
|
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11129207 |
May 13, 2005 |
|
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60513751 |
Oct 22, 2003 |
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Current U.S.
Class: |
514/220 ;
540/559 |
Current CPC
Class: |
A61P 43/00 20180101;
A61P 31/00 20180101; C07D 487/04 20130101 |
Class at
Publication: |
514/220 ;
540/559 |
International
Class: |
A61K 31/5517 20060101
A61K031/5517; A61P 43/00 20060101 A61P043/00; C07D 487/04 20060101
C07D487/04 |
Claims
1. A compound of formula I: ##STR15## or solvate thereof, wherein n
is 1 to 10, and M and M' are independently selected from monovalent
pharmaceutically acceptable cations, or together represent a
divalent pharmaceutically acceptable cation.
2. A compound according to claim 1, wherein n is 1, 3, 6, 7 or
8.
3. A compound according to claim 2, wherein n is 1 or 3.
4. A compound according to claim 1, wherein M and Mf are the
same.
5. A compound according to claim 4, wherein M and M' are alkali
metal ions.
6. A compound according to claim 5, wherein M and M' are Na+.
7. The synthesis of a compound according claim 1.
8. A compound according to claim 1 for use in a method of
therapy.
9. A pharmaceutical composition comprising a compound according to
claim 1 and a pharmaceutically acceptable excipient.
10. The use of a compound according to claim 1 in the manufacture
of a medicament for the treatment of a gene based disease.
11. A method for the treatment of a gene-based disease, comprising
administering to a subject suffering from a gene-based disease a
therapeutically-effective amount of a compound according to claim
1.
Description
[0001] This application is a continuation of U.S. patent
application Ser. No. 11/129,207, filed May 13, 2005, which is a
continuation of International Application No. PCT/GB2004/004497,
filed Oct. 22, 2004, which claims the benefit of U.S. provisional
patent application No. 60/513,751, filed Oct. 22, 2003, the
disclosures of which are incorporated by reference.
[0002] The present invention relates to bisulphite derivatives of
SJG-136 and DRG-16, and analogues thereof.
BACKGROUND TO THE INVENTION
[0003] Some pyrrolobenzodiazepines (PBDs) have the ability to
recognise and bond to specific sequences of DNA; the preferred
sequence is PuGPu. The first PBD antitumour antibiotic,
anthramycin, was discovered in 1965 (Leimgruber, et al., J. Am.
Chem. Soc., 87, 5793-5795 (1965); Leimgruber, et al., J. Am. Chem.
Soc., 87, -5791-5793 (1965)). Since then, a number of naturally
occurring PBDs have been reported, and over 10 synthetic routes
have been developed to a variety of analogues (Thurston, et al.,
Chem. Rev. 1994, 433-465 (1994)). PBDs are of the general
structure: ##STR2##
[0004] They differ in the number, type and position of
substituents, in both their aromatic A rings and pyrrolo C rings,
and in the degree of saturation of the C ring. In the B-ring there
is either an imine (N.dbd.C),a carbinolamine (NH--CH (OH)),or a
carbinolamine methyl ether (NH--CH(OMe)) at the N10-C11 position
which is the electrophilic centre responsible for alkylating DNA.
All of the known natural products have an (S)-configuration at the
chiral C11a position which provides them with a right-handed twist
when viewed from the C ring towards the A ring. This gives them the
appropriate three-dimensional shape for isohelicity within the
minor groove of B-form DNA, leading to a snug fit at the binding
site (Kohn, In Antibiotics III. Springer-Verlag, New York, pp. 3-11
(1975); Hurley and Needham-VanDevanter, Acc. Chem. Res., 19,
230-237 (1986)).Their ability to form an adduct in the minor
groove, enables them to interfere with DNA processing, hence their
use as antitumour agents.
[0005] In WO 00/12508, some of the present inventors disclosed the
following compound (SJG-136, Compound 80): ##STR3##
[0006] This compound is also disclosed in Gregson, S. J., et al.,
Chem. Commun., 1999, 797-798. It has entered Phase I clinical
trials in the UK and is likely to enter Phase I clinical trials in
the US shortly.
[0007] One difficulty that this compound presents in formulation is
that in water it converts to the di-carbinolamine form:
##STR4##
[0008] and when isolated often exists as a mixture of the imine,
mono-carbinolamine and the di-carbinolamine forms. Furthermore, if
the compound is isolated as a solid with a mixture of these three
forms, the balance between them may change over time. Although this
does not pose a problem for administration of the compound, it can
provide difficulties in accurately assessing the amount of active
substance in a given amount of powder.
[0009] SJG-136 does not appear to exhibit the cardiotoxicity that
has been associated with pyrrolobenzodiazepines in the past.
[0010] Also disclosed in WO 00/12508, is the following compound
(DRG-16, Compound 218): ##STR5##
[0011] This compound is also disclosed in Gregson, S. J., et al.,
J. Med. Chem., 2004, 1161-1174, in which it is shown to have
similar properties to SJG-136.
DISCLOSURE OF THE INVENTION
[0012] The present inventors have surprisingly discovered that the
bis-bisulphites of SJG-136 and DRG-16, and analogues thereof,
appear to be resistant to the interconversion described above, but
retain activity, and therefore are suitable for formulation.
[0013] Accordingly, in a first aspect, the invention comprises a
compound of formula I: ##STR6##
[0014] or solvate thereof, wherein n is 1 to 10, and M and M' are
independently selected from monovalent pharmaceutically acceptable
cations, or together represent a divalent pharmaceutically
acceptable cation.
[0015] When n is 1, the compound is of formula: ##STR7##
[0016] When n is 3, the compound is of formula: ##STR8##
[0017] It is preferred that n is 1, 3, 6, 7 or 8, with 1 or 3 being
more preferred.
[0018] In a second aspect, the invention comprises the synthesis of
a compound of formula I or solvate thereof.
[0019] In a third aspect, the invention comprises a compound of
formula I or solvate thereof, for use in a method of therapy.
[0020] In a fourth aspect, the invention comprises a pharmaceutical
composition comprising a compound of formula I or solvate thereof,
and a pharmaceutically acceptable excipient.
[0021] In a fifth aspect, the invention comprises the use of a
compound of formula I or solvate thereof, in the manufacture of a
medicament for the treatment of a gene-based disease.
[0022] In a sixth aspect, the invention comprises a method for the
treatment of a gene-based disease, comprising administering to a
subject suffering from a gene-based disease a
therapeutically-effective amount of a compound of formula I or
solvate thereof.
DEFINITIONS
[0023] Pharmaceutically Acceptable Cations
[0024] Examples of pharmaceutically acceptable monovalent and
divalent cations are discussed in Berge, et al., J. Pharm. Sci.,
66, 1-19 (1977), which is incorporated herein by reference.
[0025] The pharmaceutically acceptable cation may be inorganic or
organic.
[0026] Examples of pharmaceutically acceptable monovalent inorganic
cations include, but are not limited to, alkali metal ions such as
Na.sup.+ and K.sup.+. Examples of pharmaceutically acceptable
divalent inorganic cations include, but are not limited to,
alkaline earth cations such as Ca.sup.2+ and Mg.sup.2+. Examples of
pharmaceutically acceptable organic cations include, but are not
limited to, ammonium ion (i.e. NH.sub.4.sup.+) and substituted
ammonium ions (e.g. NH.sub.3R.sup.+, NH.sub.2R.sub.2.sup.+,
NHR.sub.3.sup.+, NR.sub.4.sup.+). Examples of some suitable
substituted ammonium ions are those derived from: ethylamine,
diethylamine, dicyclohexylamine, triethylamine, butylamine,
ethylenediamine, ethanolamine, diethanolamine, piperazine,
benzylamine, phenylbenzylamine, choline, meglumine, and
tromethamine, as well as amino acids, such as lysine and arginine.
An example of a common quaternary ammonium ion is N
(CH.sub.3).sub.4.sup.+.
[0027] Gene-Based Diseases
[0028] Gene-based diseases include, and are preferably,
proliferative diseases, and also include Alzheimer's disease and
bacterial, parasitic and viral infections. Any condition which may
be treated by the regulation of gene expression may be treated the
compounds of the present invention.
[0029] Proliferative Diseases
[0030] One of ordinary skill in the art is readily able to
determine whether or not a candidate compound treats a
proliferative condition for any particular cell type. For example,
assays which may conveniently be used to assess the activity
offered by a particular compound are described in the examples
below.
[0031] The term "proliferative disease" pertains to 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.
[0032] Examples of proliferative conditions include, but are not
limited to, benign, pre-malignant, and malignant cellular
proliferation, including but not limited to, neoplasms and tumours
(e.g. histocytoma, glioma, astrocyoma, osteoma), cancers (e.g. lung
cancer, small cell lung cancer, gastrointestinal cancer, bowel
cancer, colon cancer, breast carcinoma, ovarian carcinoma, prostate
cancer, testicular cancer, liver cancer, kidney cancer, bladder
cancer, pancreas cancer, brain cancer, sarcoma, osteosarcoma,
Karposi's sarcoma, melanoma), leukaemias, psoriasis, bone diseases,
fibroproliferative disorders (e.g. of connective tissues), and
atherosclerosis.
[0033] Any type of cell may be treated, including but not limited
to, lung, gastrointestinal (including, e.g. bowel, colon), breast
(mammary), ovarian, prostate, liver (hepatic), kidney (renal),
bladder, pancreas, brain, and skin.
[0034] Methods of Treatment
[0035] As described above, the present invention provides the use
of a compound of formula I in a method of therapy. Also provided is
a method of treatment, comprising administering to a subject in
need of treatment a therapeutically-effective amount of a compound
of formula I, preferably in the form of a pharmaceutical
composition, which is the third aspect of the present invention.
The term "therapeutically effective amount" is an amount sufficient
to show benefit to a patient. Such benefit may be at least
amelioration of at least one symptom. The actual amount
administered, and rate and time-course of administration, will
depend on the nature and severity of what is being treated.
Prescription of treatment, e.g. decisions on dosage, is within the
responsibility of general practitioners and other medical
doctors.
[0036] A compound may be administered alone or in combination with
other treatments, either simultaneously or sequentially dependent
upon the condition to be treated. Examples of treatments and
therapies include, but are not limited to, chemotherapy (the
administration of active agents, including, e.g. drugs); surgery;
and radiation therapy.
[0037] Pharmaceutical compositions according to the present
invention, and for use in accordance with the present invention,
may comprise, in addition to the active ingredient, i.e. a compound
of formula I, a pharmaceutically acceptable excipient, carrier,
buffer, stabiliser or other materials well known to those skilled
in the art. Such materials should be non-toxic and should not
interfere with the efficacy of the active ingredient. The precise
nature of the carrier or other material will depend on the route of
administration, which may be oral, or by injection, e.g. cutaneous,
subcutaneous, or intravenous.
[0038] Pharmaceutical compositions for oral administration may be
in tablet, capsule, powder or liquid form. A tablet may comprise a
solid carrier or an adjuvant. Liquid pharmaceutical compositions
generally comprise a liquid carrier such as water, petroleum,
animal or vegetable oils, mineral oil or synthetic oil.
Physiological saline solution, dextrose or other saccharide
solution or glycols such as ethylene glycol, propylene glycol or
polyethylene glycol may be included. A capsule may comprise a solid
carrier such a gelatin.
[0039] For intravenous, cutaneous or subcutaneous injection, or
injection at the site of affliction, the active ingredient will be
in the form of a parenterally acceptable aqueous solution which is
pyrogen-free and has suitable pH, isotonicity and stability. Those
of relevant skill in the art are well able to prepare suitable
solutions using, for example, isotonic vehicles such as Sodium
Chloride Injection, Ringer's Injection, Lactated Ringer's
Injection. Preservatives, stabilisers, buffers, antioxidants and/or
other additives may be included, as required.
[0040] Includes Other Forms
[0041] Included in the above are the ionic forms of the compound of
formula I.
[0042] In particular, a reference to the bisulphite group
(--SO.sub.3M) also includes the anionic form (--SO.sub.3.sup.-), or
solvate thereof, as well as conventional protected forms.
[0043] Isomers and Solvates
[0044] Certain compounds may exist in one or more particular
geometric, optical, enantiomeric, diastereomeric, epimeric,
atropic, stereoisomeric, tautomeric, conformational, or anomeric
forms, including but not limited to, cis- and trans-forms; E- and
Z-forms; c-, t-, and r-forms; endo- and exo-forms; R-, S-, and
meso-forms; D- and L-forms; d- and 1-forms; (+) and (-) forms;
keto-, enol-, and enolate-forms; syn- and anti-forms; synclinal-
and anticlinal-forms; .alpha.- and .beta.-forms; axial and
equatorial forms; boat-, chair-, twist-, envelope-, and
halfchair-forms; and combinations thereof, hereinafter collectively
referred to as "isomers" (or "isomeric forms").
[0045] Preferably compounds of the present invention have the
following stereochemistry at the C11 position: ##STR9##
[0046] Note that, except as discussed below for tautomeric forms,
specifically excluded from the term "isomers", as used herein, are
structural (or constitutional) isomers (i.e. isomers which differ
in the connections between atoms rather than merely by the position
of atoms in space). For example, a reference to a methoxy group,
--OCH.sub.3, is not to be construed as a reference to its
structural isomer, a hydroxymethyl group, --CH.sub.2OH. Similarly,
a reference to ortho-chlorophenyl is not to be construed as a
reference to its structural isomer, meta-chlorophenyl. However, a
reference to a class of structures may well include structurally
isomeric forms falling within that class (e.g. C.sub.1-7 alkyl
includes n-propyl and iso-propyl; butyl includes n-, iso-, sec-,
and tert-butyl; methoxyphenyl includes ortho-, meta-, and
para-methoxyphenyl).
[0047] Note that specifically included in the term "isomer" are
compounds with one or more isotopic substitutions. For example, H
may be in any isotopic form, including .sup.1H, .sup.2H (D), and
.sup.3H (T); C may be in any isotopic form, including .sup.12C,
.sup.13C, and .sup.14C; O may be in any isotopic form, including
.sup.16O and .sup.18O; and the like.
[0048] Unless otherwise specified, a reference to a particular
compound includes all such isomeric forms, including (wholly or
partially) racemic and other mixtures thereof. Methods for the
preparation (e.g. asymmetric synthesis) and separation (e.g.
fractional crystallisation and chromatographic means) of such
isomeric forms are either known in the art or are readily obtained
by adapting the methods taught herein, or known methods, in a known
manner.
[0049] It may be convenient or desirable to prepare, purify, and/or
handle a corresponding solvate of the active compound. The term
"solvate" is used herein in the conventional sense to refer to a
complex of solute (e.g. active compound, salt of active compound)
and solvent. If the solvent is water, the solvate may be
conveniently referred to as a hydrate, for example, a mono-hydrate,
a di-hydrate, a tri-hydrate, etc.
[0050] General Synthetic Routes
[0051] The compounds of formula I may be made directly from
SJG-136, whose synthesis by two alternative routes is described in
WO 00/12508, or from DRG-16, whose synthesis is also described in
WO 00/12508, or their analogues, as appropriate.
[0052] The synthesis of compound I involves the addition of a
solution of the appropriate bisulphite salt to a solution of
SJG-136 or DRG-16, or their analogues, which is usually followed by
a purification step.
[0053] Further Preferences
[0054] It is preferred that M and M' are the same and that there
are alkali metal ions, most preferably Na.sup.+.
[0055] It is preferred that the compound of formula I is present in
purified or isolated form. For the purposes of the present
invention, the term "isolated" as used herein is defined as having
been removed from its natural environment. The term "purified" as
used herein refers to having been increased in purity, wherein
"purity" is a relative term, and not to be construed as absolute
purity. The terms "isolated" and "purified" do not necessarily
imply 100% or complete isolation or purity. Rather, there are
varying degrees of isolation or purity of which one of ordinary
skill in the art recognizes as having a potential benefit or
prophylactic or therapeutic effect. In this regard, the compounds
of the present invention can be of any level of isolation or
purity. Preferably, the compound of formula I is substantially
isolated or substantially pure, such that the compounds are
substantially free of any impurities or any materials or agents
that, for example, interfere with the activity of the compounds or
make the compounds toxic.
[0056] Further preference are that the compound of formula I is
substantially free of any of the parent compound, e.g. SJG-136 or
DRG-16 or their analogues, wherein the amount of the parent
compound is preferably less than 5% by weight, and more preferably
less than 1%, 0.1% or even 0.01% by weight.
[0057] As regards the stereochemistry of the compound of formula I,
it is preferred that it comprises at least 70%, 80%, 90%, 95% or
99% in the preferred configuration for C11 as discussed above.
EXAMPLES
Example 1
Synthesis of
1,1'[[(Propane-1,3-diyl)dioxy]bis(11aS)-11-sulpho-7-methoxy-2-methylidene-
-1,2,3,10,11,11a-hexahydro-5H-pyrrolo[2,1-c][1,4-benzodiazepin-5-one]]sodi-
um salt (SJG-720)
[0058] ##STR10##
[0059] A solution of sodium bisulphite (13.2 mg, 0.127 mmol) in
water (5.2 mL) was added to a stirred solution of SJG-136 (35.19
mg, 63.3 .mu.mol) in dichloromethane (2.5 mL, Aldrich sure seal
grade). The reaction mixture was allowed to stir vigorously for 24
hours, after which time the organic and aqueous layers were
separated. TLC analysis (eluent--95:5 v/v CHCl.sub.3/MeOH) of the
aqueous phase revealed absence of SJG-136 (R.sub.f.about.0.3) and
presence of baseline material with strong uv absorption. The
aqueous layer was lyophilised to provide the bisulphite adduct
SJG-720 as a lightweight white solid (40.88 mg, 85%). Mpt.
213-216.degree. C. [.alpha.].sup.29.sub.D+126.2.degree. (c=0.0317
MeOH)
Example 2
Synthesis of
1,1'[[(Pentane-1,3-diyl)dioxy]bis(11aS)-11-sulpho-7-methoxy-2-methylidene-
-1,2,3,10,11,11a-hexahydro-5H-pyrrolo[2,1-c][1,4-benzodiazepin-5-one]]sodi-
um salt (SJG-738)
[0060] ##STR11##
[0061] A solution of sodium bisulphite (16.46 mg, 0.158 mmol) in
water (3.0 mL) was added to a stirred solution of DRG-16 (46.2 mg,
79.1 .mu.mol) in dichloromethane (1.5 mL, Aldrich sure seal grade).
The reaction mixture was allowed to stir vigorously for 22 hours,
after which time the organic and aqueous layers were separated. TLC
analysis (eluent--95:5 v/v CHCl.sub.3/MeOH) of the aqueous phase
revealed absence of DRG-16 (R.sub.f.about.0.3) and presence of
baseline material with strong uv absorption. The aqueous layer was
lyophilised to provide the bisulphite adduct SJG-738 as a
lightweight white solid (47.95 mg, 77%). Mpt. 203-204.degree. C.
[.alpha.].sup.27.sub.D+117.6.degree. (c=0.0425 MeOH).
[0062] Further Studies
[0063] All NMR experiments were performed using a Bruker Avance 400
MHz NMR spectrophotometer. NMR solvents were purchased from Goss
Scientific.
[0064] Techniques
[0065] One Dimensional NMR Experiments (Studies 1, 2 and 3)
[0066] Samples of SJG-720 (.about.3 mg) were dissolved in
d.sub.6-DMSO, D.sub.2O and d.sub.4-MeOH (Cambridge Isotopes) and
transferred to separate NMR tubes (Wilmad, 5 mm). .sup.1H NMR
spectra were obtained on a Bruker Avance 400 MHz NMR
spectrophotometer (typically 64 scans). .sup.13C and DEPT
(Distortionless Enhancement through Polarized Transfer) experiments
were performed on these samples using the same instrument at the
appropriate field strength (100 MHz).
[0067] Study 4
[0068] NMR spectra were recorded, as described above, after the
following time intervals: [0069] d.sub.6-DMSO, 1 hr, 8 days. [0070]
D.sub.2O, 3 hrs, 8 days. [0071] d.sub.4-MeOH, 10 minutes, 3
days.
[0072] Additional Studies
[0073] Standard two dimensional NMR techniques COSY (COrrelated
SpectroscopY) and HMQC (Heteronuclear Multiple Quantum Coherence)
were employed to assign NMR shifts. Comparative NMR spectra for
SJG-136 were obtained on a Bruker Avance 500 MHz NMR
instrument.
[0074] Details of Studies
[0075] Study 1: NMR Studies in d.sub.6-DMSO
[0076] A solution of SJG-720 in d.sub.6-DMSO was analysed by
.sup.1H and .sup.13C NMR spectroscopy and signals assigned (see
below) with the aid of additional NMR experiments (DEPT, COSY and
HMQC). Without wishing to be bound by theory, initial observations
suggest that the molecule exists as predominantly one
diastereoisomer and the presence of the N10-C11 imine form is not
detected. Key signals in the .sup.1H NMR include N10-H (.delta.
5.06 ppm), H11 (.delta. 3.74 ppm, d, J=10.5 Hz) and a broad singlet
at .delta. 5.06 ppm corresponding to C2.dbd.CH.sub.2. The .sup.13C
NMR DEPT spectrum also exhibits diagnostic signals such as C11
(.delta. 78.8 ppm) and the intact C2.dbd.CH.sub.2 (.delta. 107.8
ppm). ##STR12##
[0077] Assignment of NMR Signals for SJG-720 in d.sub.6-DMSO.
[0078] Study 2: NMR Studies in D.sub.2O
[0079] A solution of SJG-720 in D.sub.2O was analysed by .sup.1H
NMR spectroscopy and key signals assigned with the aid of a COSY
experiment. Although the spectrum appears relatively clean and
shows, without wishing to be bound by theory, the presence of
predominantly one diastereoisomer a number of key signals overlap
with each other, signals for H11, H11a, H3 and
OCH.sub.2CH.sub.2CH.sub.2O all resonate between .delta. 4.12-4.45
ppm. Clearly resolved signals include H6 (.delta. 7.18 ppm), H9
(.delta. 6.78 ppm), C2.dbd.CH.sub.2 (.delta. 5.27 ppm), OCH.sub.3
(.delta. 3.85 ppm), H1 (.delta. 3.08 ppm) and
OCH.sub.2CH.sub.2CH.sub.2O (distorted p, .delta. 2.36 ppm). The
exchangeable NH signal is absent from this spectrum.
[0080] Study 3: NMR Studies in d.sub.4-MeOH
[0081] NMR experiments were also performed on a solution of SJG-720
in d.sub.4-MeOH. Without wishing to be bound by theory, the .sup.1H
NMR spectrum shows the presence of only one diastereoisomer with
signals for H11, H11a, H3 and OCH.sub.2CH.sub.2CH.sub.2O
overlapping with each other. However, HMQC and COSY experiments
were able to assist in identifying H11 (.delta. 4.13 ppm, d, J=10.7
Hz). Other key signals visible in the spectrum include H6 (.delta.
7.15 ppm), H9 (.delta. 6.72 ppm), C2.dbd.CH.sub.2 (.delta. 5.16,
5.18 ppm), OCH.sub.3 (.delta. 3.83 ppm), H1 (.delta. 3.08 ppm, d,
J=16 Hz and .delta. 3.07-2.98 ppm) and OCH.sub.2CH.sub.2CH.sub.2O
(p, .delta. 2.33 ppm, J=6.1 Hz). The exchangeable NH signal is
absent from this spectrum. Key signals in the .sup.1H NMR spectrum
correlate (HMQC) with the corresponding carbon signals in the
.sup.13C DEPT NMR: C6 (.delta. 113.6 ppm), C2.dbd.CH.sub.2 (.delta.
109.4 ppm), C9 (.delta. 108.5 ppm), C11 (.delta. 81.5 ppm),
OCH.sub.2CH.sub.2CH.sub.2O (.delta. 66.6 ppm), C11a (.delta. 58.9
ppm), OCH.sub.3 (.delta. 57.0 ppm), C3 (.delta. 51.6 ppm), C1
(.delta. 37.1 ppm) and OCH.sub.2CH.sub.2CH.sub.2O (.delta. 30.2
ppm).
[0082] Study 4: Stability in d.sub.6-DMSO, d.sub.4-MeOH and
D.sub.2O
[0083] As discussed above, PBDs exist in three interconvertable
forms around the N10-C11 bond and this is shown for SJG-136:
##STR13##
[0084] NMR experiments were carried out on SJG-136 in its
carbinolamine forms. The carbinolamine forms are revert to the
imine form immediately if analysed in either CDCl.sub.3 or
CD.sub.3CN. However, strong signals corresponding to the kinetic
and thermodynamic carbinolamine forms can be observed when the
sample is analysed in d.sub.6-DMSO. These gradually disappear from
the spectra over a period of about 24 hours when the imine form
becomes the predominant species.
[0085] Samples of the bisulphite adduct SJG-720 dissolved in
d.sub.6-DMSO and D.sub.2O were stored at room temperature for over
a period of 7 days and then analysed by .sup.1H NMR spectroscopy.
In the case of the D.sub.2O solution the spectrum remained
unchanged from day 1, i.e. no appreciable hydrolysis had occurred.
Similarly, the spectrum obtained from prolonged storage of the
sample in d.sub.6-DMSO showed the complete absence of parent imine
SJG-136 signals. In addition, a sample of SJG-720 stored at room
temperature in d.sub.4-MeOH for over 3 days was also analysed by
.sup.1H NMR. Again, interconversion to the carbinolamine methyl
ether from was not observed by NMR.
[0086] Study 5: Stereochemistry at C11
[0087] The NMR studies described above appear to have determined
that the PBD bisulphite adducts are formed in a stereocontrolled
fashion to afford almost exclusively a single diastereomeric
product.
[0088] NMR studies of SJG-720 in d.sub.4-MeOH reveal a coupling
constant of .about.10 Hz between the vicinal protons H11 and H11a.
This coupling constant is consistent with a large dihedral angle
and trans stereochemistry between the vicinal protons. It therefore
appears that SJG-720 possesses the absolute stereochemistry shown
below: ##STR14##
[0089] Study 6: NMR spectra of SJG-738
[0090] .sup.1H-NMR (400 MHz, d.sub.6 DMSO) .delta. 7.00 (s, 2H),
6.43 (s, 2H), 5.08 (s, 4H), 5.04 (s, 2H), 4.22 (d, J=15.78 Hz),
4.03-3.89 (m, 8H), 3.71 (s, 6H), 3.71 (d, J=10.5 Hz, 2H), 3.23 (d,
15.7 Hz, 2H), 2.87-2.81 (dd, J=15.8, 9.1 Hz, 2H), 1.82 (p, J=7.3
Hz, 4H), 1.63-1.54 (m, 2H).
[0091] .sup.13C NMR (100 MHz, d.sub.6 DMSO) .delta. 167.1, 151.1,
143.8, 142.9, 140.1, 116.3, 112.4, 107.8, 106.3, 78.8, 68.0, 56.7,
55.9, 50.3, 35.7, 28.4, 22.2.
[0092] Biological Results
[0093] K562 Assay
[0094] An initial stock of compound (in 0.006% DMSO) was used to
generate a 1:3 dilution series ranging from 600 nM to 30 pM be the
sequential addition of 40 .mu.L compound to 80 .mu.L of solvent
(0.006% DMSO in sterile H.sub.2O). 10 .mu.L of each point of the
dilution range was transferred to a 96 well assay plate (Nunc, Cat.
No. 163320) to generate four replica points per compound
concentration. K562 human chronic myeloid leukaemia cells were
maintained in RPM1 1640 medium supplemented with 10% foetal calf
serum and 2 mM glutamine at 37.degree. C. in a humidified
atmosphere containing 5% Co.sub.2. 190 .mu.L of a 5.times.10.sup.4
cell solution was added to each well of the assay plate using a
Matrix Wellmate (Apogent Discoveries), resulting in a total of
9.5.times.10.sup.3 cells/well. Working compound concentrations
therefore ranged from 30 nM to 1.5 pM. Plates were then kept in the
dark at 37.degree. C. in a humidified atmosphere containing 5%
CO.sub.2. The assay is based on the ability of viable cells to
reduce a yellow soluble tetrazolium salt,
3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl-2H-tetrazolium bromide
(MTT, Aldrich-Sigma), to an insoluble purple formazan precipitate.
Following incubation of the plates for 4 days (to allow control
cells to increase in number by approximately 10 fold), 25 .mu.L of
MTT solution (5 mg/mL in phosphate-buffered saline) was added to
each well and the plates further incubated for 4 hours. The bulk of
the medium was then pipetted from the cell pellet leaving 10-20
.mu.L per well. DMSO (200 .mu.L) was added to each well and the
samples agitated to ensure complete mixing. The optical density was
then read at a wavelength of 540 nm on a Fusion plate reader
(Perkin Elmer). A dose-response curve was constructed using
GraphPad Prism 4.01 (GraphPad Software Inc.) from n=2 data (from 2
individual experiments). An IC.sub.50 value was read as the dose
required to reduce the final optical density to 50% of the control
value.
[0095] The IC.sub.50 values measured for SJG-720, SJG-136 and
SJG-738 were less than 10 nM.
[0096] DNA Cross-Linking
[0097] Closed-circular puc18 DNA was linearized with HindIII, then
dephosphorylated, and finally 5' end labeled with [.gamma.32P]-ATP
using polynucleotide kinase. Reactions containing 10 ng of DNA and
drug were carried out in aqueous 1.times.TEOA (25 mM
triethanolamine, 1 mM EDTA, pH 7.2) buffer at a final volume of 50
.mu.L at 37.degree. C. Reactions were terminated by addition of an
equal volume of stop solution (0.6 M NaOAc, 20 mM EDTA, 100
.mu.g/mL tRNA) followed by precipitation with ethanol. Following
centrifugation of the samples, the supernatants were discarded and
the pellets were dried by lyophilization. Samples were resuspended
in 10 .mu.L of alkaline denaturing buffer (4 mg bromophenol blue,
600 mg sucrose and 40 mg NaOH) and vortexed for three minutes at
room temperature. The non-denatured controls were re-suspended in
10 .mu.L of standard sucrose loading dye (2.5 mg bromophenol blue,
2.5 mg xylene cyanol blue and 4 g sucrose). Both samples and
controls were loaded directly onto an agarose gel.
[0098] Electrophoresis was performed on a 0.8% submerged horizontal
agarose gel, 20 cm in length for 16 hours at 38-40 V in 1.times.TAE
running buffer (2.42 g Tris Base, 0.372 g EDTA, 0.571 ml glacial
acetic acid). Gels were dried under vacuum for 80 minutes at
80.degree. C. on a Savant SG210D SpeedGel gel dryer onto one layer
of Whatman 3MM with a layer of DE81 filter paper underneath. An
autoradiograph was obtained, after overnight exposure onto FujiRX
x-ray film. The film bands were quantitated using a BioRad GS-670
imaging laser densitometer. The percentage of cross-linking was
calculated by measuring the total DNA in each lane (the sum of the
densities for the double-stranded and single-stranded bands)
relative to the amount of density of double-stranded band alone. A
dose response curve was derived by plotting drug concentration
against the determined percentage level of cross-linked DNA, from
which was derived the amount required to cross-link 50% of the DNA
(XL.sub.50)
[0099] After 2 hours the XL.sub.50 values measured for both SJG-136
and SJG-720 were less than 500 nM.
* * * * *