U.S. patent application number 13/296352 was filed with the patent office on 2012-03-08 for macrolides with anti-inflammatory activity.
This patent application is currently assigned to Glaxo Group Limited. Invention is credited to Sulejman Alihodzic, Martina Bosnar, Ognjen Culic, Vesna Erakovic Haber, Antun Hutinec, Dubravko Jelic, Goran Kragol, Nikola Marjanovic, Zorica Marusic-Istuk, Marija Ribic, Vanja Vela.
Application Number | 20120058963 13/296352 |
Document ID | / |
Family ID | 36916830 |
Filed Date | 2012-03-08 |
United States Patent
Application |
20120058963 |
Kind Code |
A1 |
Alihodzic; Sulejman ; et
al. |
March 8, 2012 |
Macrolides with Anti-Inflammatory Activity
Abstract
The present invention relates to novel semi-synthetic macrolides
having anti-inflammatory activity. More particularly, the invention
relates to 14- and 15-membered macrolides substituted at the 4''
position, to their pharmaceutically acceptable derivatives, to
processes and intermediates for their preparation, to
pharmaceutical compositions containing them and to their activity
and use in the treatment of inflammatory diseases and conditions in
humans and animals, especially those diseases associated with
excessive secretion of TNF-.alpha., IL-1, IL-8, IL-2 or IL-5;
and/or inhibitor of excessive lymphocyte proliferation; and/or
excessive granulocyte degranulation.
Inventors: |
Alihodzic; Sulejman; (Strmec
Samoborski, HR) ; Bosnar; Martina; (Zagreb, HR)
; Culic; Ognjen; (Zagreb, HR) ; Erakovic Haber;
Vesna; (Rijeka, HR) ; Hutinec; Antun; (Zagreb,
HR) ; Jelic; Dubravko; (Samobor, HR) ; Kragol;
Goran; (Zagreb, HR) ; Marjanovic; Nikola;
(Zagreb, HR) ; Marusic-Istuk; Zorica; (Samobor,
HR) ; Ribic; Marija; (Durmanec, HR) ; Vela;
Vanja; (Zagreb, HR) |
Assignee: |
Glaxo Group Limited
Greenford
GB
|
Family ID: |
36916830 |
Appl. No.: |
13/296352 |
Filed: |
November 15, 2011 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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11813873 |
Jul 13, 2007 |
8080529 |
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PCT/IB2006/001238 |
Jan 13, 2006 |
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13296352 |
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60643841 |
Jan 13, 2005 |
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60715828 |
Sep 9, 2005 |
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Current U.S.
Class: |
514/29 ; 514/28;
536/7.1; 536/7.4 |
Current CPC
Class: |
A61P 7/10 20180101; A61P
29/00 20180101; A61P 37/02 20180101; A61P 37/00 20180101; A61P
11/06 20180101; A61P 17/06 20180101; C07H 17/00 20130101; A61P
17/10 20180101; A61P 1/00 20180101; A61P 19/02 20180101; A61P 27/02
20180101; C07H 17/08 20130101; A61P 43/00 20180101; A61P 11/00
20180101; A61P 7/00 20180101; A61P 11/02 20180101; A61P 1/04
20180101 |
Class at
Publication: |
514/29 ; 536/7.1;
536/7.4; 514/28 |
International
Class: |
A61K 31/7048 20060101
A61K031/7048; C07H 17/08 20060101 C07H017/08; A61K 31/7052 20060101
A61K031/7052; A61K 31/706 20060101 A61K031/706; A61P 37/02 20060101
A61P037/02; A61P 11/06 20060101 A61P011/06; A61P 11/00 20060101
A61P011/00; A61P 1/00 20060101 A61P001/00; C07H 17/00 20060101
C07H017/00; A61P 29/00 20060101 A61P029/00 |
Claims
1. A compound of Formula (I) ##STR00152## wherein: A is a bivalent
radical selected from --C(O)--, --N(R.sup.7)CH.sub.2--,
--CH.sub.2N(R.sup.7)--, --CH(OH)-- and --C(.dbd.NOR.sup.7)--;
R.sup.1 is --OC(O)(CH.sub.2).sub.nNR.sup.8R.sup.9,
--O--(CH.sub.2).sub.nNR.sup.8R.sup.9,
--OC(O)N(R.sup.7)(CH.sub.2).sub.nNR.sup.8R.sup.9, ##STR00153##
--O(CH.sub.2).sub.nCN , --OC(O)(CH.sub.2).sub.n N
(CH.sub.2).sub.nNR.sup.8R.sup.9, or --OC(O)CH.dbd.CH.sub.2 with the
proviso that if R.sup.1 is --OC(O)CH.dbd.CH.sub.2, R.sup.3 cannot
be methyl; R.sup.2 is hydrogen or a hydroxyl protecting group;
R.sup.3 is C.sub.1-4 alkyl substituted at terminal carbon atom with
CN or NH.sub.2 group, or C.sub.1-5 alkanoyl; R.sup.4 is hydrogen,
C.sub.1-4 alkyl, or C.sub.2-6 alkenyl; R.sup.5 is hydroxy, methoxy
group, --OC(O)(CH.sub.2).sub.nNR.sup.8R.sup.9,
--O--(CH.sub.2).sub.nNR.sup.8R.sup.9, or --O(CH.sub.2).sub.nCN;
R.sup.6 is hydroxy; or R.sup.5 and R.sup.6 taken together with the
intervening atoms form a cyclic group having the following
structure: ##STR00154## wherein Y is a bivalent radical selected
from --CH.sub.2--, --CH(CN)--, --O--, --N(R.sup.7)-- and
--CH(SR.sup.7)--; R.sup.7 is hydrogen or C.sub.1-6 olkyl; R.sup.8
and R.sup.9 are each independently hydrogen, C.sub.3-7 cycloalkyl,
or C.sub.1-18 alkyl, wherein C.sub.1-18 alkyl is: (i) uninterrupted
or interrupted by 1-3 bivalent radical groups selected from --O--,
--S-- and --N(R.sup.7)--; and/or (ii) unsubstituted or substituted
by 1-3 groups selected from halogen, OH, NH.sub.2,
N--(C.sub.1-C.sub.6)alkylamino, N,N-di(C.sub.1-C.sub.6-alkyl)amino,
CN, NO.sub.2, OCH.sub.3, a C.sub.3-8 membered non-aromatic ring
which is saturated or unsaturated, a non-aromatic heterocyclic ring
containing 2-6 carbon atoms which is saturated or unsaturated
containing from 1-2 heteroatoms selected from oxygen, sulphur and
nitrogen, alkylcarbonylalkoxy and alkoxycarbonylamino; or R.sup.8
and R.sup.9 taken together with nitrogen to which they are attached
form a non-aromatic heterocyclic ring containing 2-6 carbon atoms
which is: (iii) saturated or unsaturated containing from 0 or 1
additional heteroatoms selected from oxygen, sulphur and nitrogen;
and/or (iv) unsubstituted or substituted by 1-2 groups selected
from C.sub.1-5alkanoyl and C.sub.1-8 alkyl, wherein C.sub.1-8 alkyl
is uninterrupted or is interrupted by 1-3 bivalent radical groups
selected from --O--, --S-- and --N(R.sup.7)--, and/or being
unsubstituted or substituted by 1-2 groups selected from OH,
NH.sub.2, a non-aromatic heterocyclic ring containing 2-6 carbon
atoms which is unsubstituted or is substituted by group selected
from C.sub.1-4 alkyl, halo, NH.sub.2, OH, SH, C.sub.1-6 alkoxy and
C.sub.1-4 hydroxyalkyl, a C.sub.3-7 cycloalkyl which is
unsubstituted or is substituted by group selected from C.sub.1-4
alkyl, halo, NH.sub.2, OH, SH, C.sub.1-6 alkoxy and C.sub.1-4
hydroxyalkyl; n is an integer from 1 to 8; or a pharmaceutically
acceptable salt thereof.
2. A compound of Formula (I) according to claim 1 wherein A is a
bivalent radical selected from --C(O)--, --CH(OH)-- and
--C(.dbd.NOR.sup.7)--.
3. A compound of Formula (I) according to claim 1 wherein A is a
bivalent radical selected from --N(R.sup.7)CH.sub.2-- and
--CH.sub.2N(R.sup.7)--.
4. A compound of Formula (I) according to claim 1 wherein R.sup.3
is C.sub.1-4aminoalkyl.
5. A compound of Formula (I) according to claim 1 wherein R.sup.3
is C.sub.1-4 cyanoalkyl.
6. A compound of Formula (I) according to claim 1 wherein R.sup.3
is C.sub.1-5alkanoyl.
7. A compound of Formula (I) according to claim 1 wherein R.sup.5
and R.sup.6 taken together with the intervening atoms form a cyclic
group having the following structure: ##STR00155## wherein Y is a
bivalent radical selected from --O-- and --N(R.sup.7)--.
8. A compound of Formula (I) according to claim 1, wherein R.sup.8
and R.sup.9 taken together with nitrogen to which they are attached
form a C.sub.5-7 membered saturated non-aromatic heterocyclic
ring.
9. A pharmaceutical composition comprising a compound of Formula
(I) according to claim 1 or a pharmaceutically acceptable salt
thereof and a pharmaceutically acceptable diluent or carrier.
10. A method of treating an inflammatory disease, disorder or
condition characterized by or associated with an undesirable
inflammatory immune response, or an excessive secretion of
TNF-.alpha., IL-1, IL-6 and IL-8 which comprises administering to a
subject in need thereof a therapeutically effective amount of a
compound according to claim 1.
11. A method of treating an inflammatory condition or immune or
anaphylactic disorder associated with infiltration of leukocytes
into inflamed organ or tissue comprising administering to a subject
in need thereof a therapeutically effective amount of a compound of
claim 1.
12. The method according to claim 11, wherein the inflammatory
condition or immune disorder is selected from the group consisting
of asthma, COPD, diffuse panbronchiolitis, adult respiratory
distress syndrome, inflammatory bowel disease, Crohn's disease,
chronic bronchitis, and cystic fibrosis.
13. A method according to claim 1, wherein said inflammatory
condition or immune disorder is selected from the group consisting
of an inflammatory condition or immune disorder of the lungs,
joints, eyes, bowel, skin, and heart.
14. A method according to claim 13, wherein said inflammatory
condition or immune disorder is selected from the group consisting
of asthma, adult respiratory distress syndrome, bronchitis,
bronchiectasis, bronchiolitis obliterans, cystic fibrosis,
rheumatoid arthritis, rheumatoid spondylitis, osteoarthritis,
osteomyelitis, sinusitis, nasal polyps, gouty arthritis, uveitis,
conjunctivitis, inflammatory bowel disease, Crohn's disease,
ulcerative colitis, distal proctitis, psoriasis, eczema,
dermatitis, acne, coronary infarct damage, chronic inflammation,
endotoxin shock, chronic sinusitis, pulmonary fibrosis, diffuse
panbronchiolitis, and smooth muscle proliferation disorders.
15. A method of inhibiting one or more inflammatory processes
selected from the group consisting of: proinflammatory cytokine
production, lymphocyte proliferation, granulocyte degranulation,
t-cell proliferation, neutrophilia, and oedema comprising exposing
an organ or tissue afflicted with inflammation to an amount of a
compound according to claim 1 effective to inhibit said
inflammatory process.
16. The method of claim 15, wherein the inflammatory process
comprises lymphocyte proliferation.
Description
[0001] This application claims priority to U.S. Provisional
Application No. 60/643,841 filed Jan. 13, 2005, and U.S.
Provisional Application 60/715,828 filed September 9, 2005, each of
which are incorporated herein by reference in their entirety.
FIELD OF THE INVENTION
[0002] The present invention relates to substituted macrolides
having anti-inflammatory activity, their pharmaceutically
acceptable derivatives, and to methods of use thereof.
TECHNICAL PROBLEM
[0003] The invention is directed to solving the technical problem
of providing novel targeted anti-inflammatory agents. More
specifically, the invention provides anti-inflammatory agents
wherein the active substance is neither a steroid nor an NSAID. The
compounds of the invention are responsive to this problem by virtue
of their anti-inflammatory activity and their ability to accumulate
in various immune cells recruited to the locus of inflammation.
BACKGROUND OF THE INVENTION
[0004] Inflammation is the final common pathway of various insults,
such as infection, trauma, allergies to the human body. It is
characterized by activation of the immune system with recruitment
of inflammatory cells, production of pro-inflammatory cells and
production of pro-inflammatory cytokines.
[0005] Most inflammatory diseases are characterized by abnormal
accumulation of inflammatory cells including monocytes/macrophages,
granulocytes, plasma cells, lymphocytes and platelets. Along with
tissue endothelial cells and fibroblasts, these inflammatory cells
release a complex array of lipids, growth factors, cytokines and
destructive enzymes that cause local tissue damage.
[0006] One form of inflammatory response is neutrophilic
inflammation which is characterized by infiltration of the inflamed
tissue by neutrophil polymorphonuclear leucocytes (PMN), which are
a major component of host defence. Tissue infection by
extracellular bacteria represents the prototype of this
inflammatory response. On the other hand, various non-infectious
diseases are characterized by extravascular recruitment of
neutrophils. This group of inflammatory diseases includes chronic
obstructive pulmonary disease, adult respiratory distress syndrome,
some types of immune-complex alveolitis, cystic fibrosis,
bronchitis, bronchiectasis, emphysema, glomerulonephritis,
rheumatoid arthritis, gouty arthritis, ulcerative colitis, certain
dermatoses such as psoriasis and vasculitis. In these conditions
neutrophils are thought to play a crucial role in the development
of tissue injury which, when persistent, can lead to the
irreversible destruction of the normal tissue architecture with
consequent organ dysfunction. Tissue damage is primarily caused by
the activation of neutrophils followed by their release of
proteinases and increased production of oxygen species.
[0007] Chronic obstructive pulmonary disease (COPD) is described by
the progressive development of airflow limitation that is not fully
reversible (ATC, 1995). Most patients with COPD have three
pathological conditions: bronchitis, emphysema and mucus plugging.
This disease is characterized by a slowly progressive and
irreversible decrease in forced expiratory volume in the first
second of expiration (FEV.sub.1), with relative preservation of
forced vital capacity (FVC) (Barnes, N. Engl. J. Med. (2000),
343(4): 269-280). In both asthma and COPD there is significant, but
distinct, remodeling of airways. Most of the airflow obstruction is
due to two major components, alveolar destruction (emphysema) and
small airways obstruction (chronic obstructive bronchitis). COPD is
mainly characterized by profound mucus cell hyperplasia.
[0008] Cigarette smoking, air pollution and other environmental
factors are major causes of the disease. The causal mechanism
remains currently undefined but oxidant-antioxidant disturbances
are strongly implicated in the development of the disease. COPD is
a chronic inflammatory process that differs markedly from that seen
in asthma, with different inflammatory cells, mediators,
inflammatory effects, and responses to treatment (Keatings et al.,
Am. J. Respir. Crit. Care Med. (1996), 153: 530-534). Neutrophil
infiltration of the patient's lungs is a primary characteristic of
COPD.
[0009] Elevated levels of proinflammatory cytokines like
TNF-.alpha., and especially chemokines like IL-8 and GRO-a play a
very important role in pathogenesis of this disease. Platelet
thromboxane synthesis is also enhanced in patients with COPD
(Keatings et al., Am. J. Respir. Grit. Care Med. (1996), 153:
530-534; Stockley and Hill, Thorax (2000), 55(7): 629-630). Most of
the tissue damage is caused by activation of neutrophils followed
by their release of (metallo)proteinases, and increased production
of oxygen species (Repine et al., Am. J. Respir. Grit. Care Med.
(1997), 156: 341-357; Barnes, Chest (2000), 117(2 Suppl):
10S-14S).
[0010] Most therapeutic endeavour is directed towards the control
of symptoms (Barnes, Trends Pharm. Sci. (1998), 19(10): 415-423;
Barnes, Am. J. Respir. Crit. Care Med. (1999) 160: S72-S79; Hansel
et al., Expert Opin. Investig. Drugs (2000) 9(1): 3-23). Symptoms
usually equate with airflow limitation and bronchodilators are the
conventional therapy of choice. Prevention and treatment of
complications, prevention of deterioration and improved quality and
length of life are also primary goals stated in the three key
international guidelines for the management of COPD (Culpitt and
Rogers, Exp. Opin. Pharmacother. (2000) 1(5): 1007-1020; Hay, Curr.
Opin. Chem. Biol. (2000), 4: 412-419). Basically, most of the
current therapeutic research has been focused on mediators involved
in the recruitment and activation of neutrophils, or attenuation of
consequences of their undesirable activation (Stockley et al.,
Chest (2000), 117(2 Suppl): 58S-62S).
[0011] In 1975, TNF-.alpha. was defined as an endotoxin-induced
serum factor causing tumor necrosis in vitro and in vivo (Carswell
E. A. et al. Proc. Natl. Acad. Sci. U.S.A. 1975, 72, 3666-3670). In
addition to antitumor activity, TNF-.alpha. has several other
biologic activities that are important in homeostasis as well as in
pathophysiological conditions. The main sources of TNF-.alpha. are
monocytes-macrophages, T-lymphocytes and mast cells.
[0012] The finding that anti-TNF-.alpha. antibodies (cA2) are
effective in the treatment of patients suffering from rheumatoid
arthritis (RA) (Elliot M. et al. Lancet 1994, 344, 1105-1110)
intensified the interest to find new TNF-.alpha. inhibitors as
possible potent medicaments for RA. Rheumatoid arthritis is an
autoimmune chronic inflammatory disease characterized by
irreversible pathological changes of the joints. In addition to RA,
TNF-.alpha. antagonists are also applicable to several other
pathological conditions and diseases such as spondylitis,
osteoarthritis, gout and other arthritic conditions, sepsis, septic
shock, toxic shock syndrome, atopic dermatitis, contact dermatitis,
psoriasis, glomerulonephritis, lupus erhythematosus, scleroderma,
asthma, cachexia, chronic obstructive lung disease, congestive
heart failure, insulin resistance, lung fibrosis, multiple
sclerosis, Crohn's disease, ulcerative colitis, viral infections
and AIDS.
[0013] The interest of the scientific community has recently turned
towards the immunomodulating and anti-inflammatory activities of
the macrolide antibiotics (Journal of Antimicrobial Chemotherapy,
1988, 41, Suppl. B, 37-46).
[0014] An ideal immunomodulating agent should be able to suppress
the deleterious effects of the inflammatory response, while leaving
the protective immune responses intact.
[0015] Macrolide antibiotics accumulate preferentially within
different cells of subjects, especially within phagocyte cells such
as mononuclear peripheral blood cells, and peritoneal and alveolar
macrophages. (Gladue, R. P. et al, Antimicrob. Agents Chemother.
1989, 33, 277-282; Olsen, K. M. et al, Antimicrob. Agents
Chemother. 1996, 40, 2582-2585). Anti-inflammatory effects of some
macrolides have been described in the literature. For example, the
anti-inflammatory effect of erythromycin derivatives (J.
Antimicrob. Chemother. 1998, 41, 37-46; WO Patent Application No.
00/42055). Taisho claims further anti-inflammatory erythromycin
derivatives modified in positions 3, 9, 11 and 12 (EP 0775489 and
EP 0771564). In the patent application WO 02/087596, there is a
good description of the anti-inflammatory activity of azithromycin,
a known antibacterial agent. Azithromycin derivatives lacking the
sugar moieties cladinose and desosamine and having
anti-inflammatory activity have been described (Pliva, U.S. Oat.
No. 4,886,792). International patent applications WO 04/039821 and
WO 04/013153 (Zambon Group) disclose macrolide and azalide
derivatives lacking cladinose sugar that exhibit anti-inflammatory
but not antibacterial activity.
[0016] Anti-inflammatory effects of some macrolides are also known
from in vitro and in vivo studies in experimental animal models
such as in zymosan-induced peritonitis in mice (J. Antimicrob.
Chemother. 1992, 30, 339-348) and endotoxin-induced neutrophil
accumulation in rat trachea (J. Immunol. 1997, 159, 3395-4005). The
modulating effect of macrolides upon cytokines such as interleukin
8 (IL-8) (Am. J. Respir. Grit. Care. Med. 1997, 156, 266-271) and
interleukin 5 (IL-5) (EP Pat. No. 0775489 and EP Pat. No. 771564)
is known as well.
[0017] Macrolides have proved to be useful in the treatment of
inflammatory pathologies such as panbronchiolitis (Thorax, 1997,
52, 915-918), bronchial asthma (Chest, 1991, 99 670-673), and
azithromycin in particular has proved effective in improving lung
function in patients with cystic fibrosis (The Lancet, 1998, 351,
420).
[0018] The administration of macrolides to asthmatics is
accompanied by a reduction in hypersecretion and in bronchial
hypersensitivity resulting from the macrolides, anti-oxidative and
anti-inflammatory interaction with phagocytes and in particular
with neutrophilis (Inflammation, Vol. 20, No. 6, 1996).
SUMMARY OF THE INVENTION
[0019] New 14- and 15-membered macrolide compounds substituted at
the 4'' position of cladinose sugar moiety, represented by the
Formula (I), representing the subject of the present invention,
their pharmaceutically acceptable derivatives and pharmaceutical
compositions comprising them have hitherto not been described.
Moreover, no compound representing the subject of the present
invention has been described either as an anti-inflammatory
substance or as an inhibitor of one or more of TNF-.alpha., IL-1
(IL-1.alpha. or IL-1.beta.), IL-6, IL-8, IL-2 or IL-5; and/or
inhibitor of excessive lymphocyte proliferation; and/or excessive
granulocyte degranulation. Consequently, the use of such compounds
to combat inflammatory states has not been described or suggested.
Nor has there been a description or suggestion of pharmaceutical
dosage forms containing effective amounts of a 14- and 15-membered
macrolide compounds substituted at the 4'' position for treating
inflammatory states in a mammalian subject, including a human.
[0020] A characteristic of compounds represented by Formula (I) is
selective accumulation in target organs and cells in the above
mentioned inflammatory diseases and conditions. These
pharmacokinetic properties enable the compounds represented by
Formula I to act at the inflammation site in inflammation cells by
inhibiting the production of inflammation mediators. In such a
manner, the unfavourable systemic side-effects that are
characteristic of corticosteroids or non-steroidal
anti-inflammatory molecules are avoided and the therapeutic action
of compounds represented by Formula (I) is targeted to the area
where it is most needed. Following local or systemic application,
molecules rapidly accumulate in inflammation cells wherein they act
by inhibiting the production of cytokines and chemokines and/or
other inflammatory mediators thus suppressing the inflammation.
[0021] Thus, the present invention is directed to: [0022] (a)
compounds Represented by Formula (I)
##STR00001##
[0022] wherein [0023] A is a bivalent radical selected from
--C(O)--, --NHC(O)--, --C(O)NH--, --N(R.sup.7)CH.sub.2--,
--CH.sub.2N(R.sup.7)--, --CH(OH)-- and --C(.dbd.NOR.sup.7)--;
[0024] R.sup.1 is --OC(O)(CH.sub.2).sub.nNR.sup.8R.sup.9,
--O--(CH.sub.2).sub.nNR.sup.8R.sup.9,
--OC(O)N(R.sup.7)(CH.sub.2).sub.nNR.sup.8R.sup.9,
##STR00002##
[0024] --O(CH.sub.2).sub.nCN,
--OC(O)(CH.sub.2).sub.nN(CH.sub.2).sub.nNR.sup.8R.sup.9, or
--OC(O)CH.dbd.CH.sub.2 with the proviso that if R.sup.1 is
--OC(O)CH.dbd.CH.sub.2, R.sup.3 cannot be methyl; [0025] R.sup.2 is
hydrogen or a hydroxyl protecting group; [0026] R.sup.3 is
hydrogen, unsubstituted C.sub.1-4 alkyl or C.sub.1-4 alkyl
substituted at terminal carbon atom with CN or NH.sub.2 group, or
C.sub.1-5 alkanoyl; [0027] R.sup.4 is hydrogen, C.sub.1-4 alkyl or
C.sub.2-6 alkenyl or a hydroxyl protecting group 1; [0028] R.sup.5
is hydroxy, methoxy group, --OC(O)(CH.sub.2).sub.nNR.sup.8R.sup.9
--O--(CH.sub.2).sub.nNR.sup.8R.sup.9 or O(CH.sub.2).sub.nCN; [0029]
R.sup.6 is hydroxy; or [0030] R.sup.5 and R.sup.6 taken together
with the intervening atoms form a cyclic group having the following
structure:
##STR00003##
[0030] wherein Y is a bivalent radical selected from --CH.sub.2--,
--CH(CN)--, --O--, --N(R.sup.7)-- and --CH(SR.sup.7)--; [0031]
R.sup.7 is hydrogen or C.sub.1-6alkyl; [0032] R.sup.8 and R.sup.9
are each independently hydrogen, C.sub.3-7 cycloalkyl, C.sub.1-18
alkyl, wherein C.sub.1-18 alkyl is: [0033] (i) uninterrupted or
interrupted by 1-3 bivalent radical groups selected from --O--,
--S-- and --N(R.sup.7)--; and/or [0034] (ii) unsubstituted or
substituted by 1-3 groups selected from halogen, OH, NH.sub.2,
N--(C.sub.1-C.sub.6)alkylamino (preferably N-methylamino or
N-ethylamino), N,N-di(C.sub.1-C.sub.6-alkyl)amino (preferably
dimethylamino, diethylamino or di-isopropylamino), CN, NO.sub.2,
OCH.sub.3, a C.sub.3-8 membered non-aromatic ring which is
saturated or unsaturated a non-aromatic heterocyclic ring
containing 2-6 carbon atoms which is saturated or unsaturated
containing from 1-2 heteroatoms selected from oxygen, sulphur and
nitrogen, alkylcarbonylalkoxy and alkoxycarbonylamino; or [0035]
R.sup.8 and R.sup.9 taken together with nitrogen to which they are
attached form a non-aromatic heterocyclic ring containing 2-6
carbon atoms which is: [0036] i) saturated or unsaturated
containing from 0 or 1 additional heteroatoms selected from oxygen,
sulphur and nitrogen; and/or [0037] ii) unsubstituted or
substituted by 1-2 groups selected from C.sub.1-5alkanoyl and
C.sub.1-6alkyl, wherein C.sub.1-6alkyl is uninterrupted or is
interrupted by 1-3 bivalent radical groups selected from --O--,
--S-- and --N(R.sup.7)--, and/or being unsubstituted or substituted
by 1-2 groups selected from OH, NH.sub.2, a non-aromatic
heterocyclic ring containing 2-6 carbon atoms which is
unsubstituted or is substituted by group selected from C.sub.1-4
alkyl, halo, NH.sub.2, OH, SH, C.sub.1-6alkoxy and C.sub.1-4
hydroxyalkyl, a C.sub.3-7 cycloalkyl which is unsubstituted or is
substituted by group selected from C.sub.1-4 alkyl, halo, NH.sub.2,
OH, SH, C.sub.1-6 alkoxy and C.sub.1-4 hydroxyalkyl; [0038] n is an
integer from 1 to 8; [0039] and pharmaceutically acceptable
derivatives of the Formula I compounds; [0040] (b) compositions
containing one or more of the foregoing compounds in an amount
effective to reduce inflammation and thereby treat disorders and
conditions involving inflammation in mammals, including humans;
[0041] (c) methods for using these compounds to treat such
disorders and conditions, uses of these compounds in the treatment
of such disorders and conditions and in the preparation of
medicaments for that purpose; and [0042] (d) methods of inhibiting
inflammatory process such as proinflammatory cytokine production,
excessive lymphocyte proliferation, and excessive granulocyte
degranulation, t-cell proliferation, immune response to an antigen,
neutrophilia, or oedema
DETAILED DESCRIPTION OF THE INVENTION
[0043] The phrase "pharmaceutically acceptable", as used in
connection with compositions of the invention, refers to molecular
entities and other ingredients of such compositions that are
physiologically tolerable and do not typically produce untoward
reactions when administered to a mammal (e.g., human). Preferably,
as used herein, the term "pharmaceutically acceptable" means
approved by a regulatory agency of the Federal or a state
government or listed in the U.S. Pharmacopeia or other generally
recognized pharmacopeia for use in mammals, and more particularly
in humans.
[0044] The term "carrier" applied to pharmaceutical compositions of
the invention refers to a diluent, excipient, or vehicle with which
an active compound is administered. Such pharmaceutical carriers
can be sterile liquids, such as water, saline solutions, aqueous
dextrose solutions, aqueous glycerol solutions, and oils, including
those of petroleum, animal, vegetable or synthetic origin, such as
peanut oil, soybean oil, mineral oil, sesame oil and the like.
However, since the macrolides are highly soluble, aqueous solutions
are preferred. Suitable pharmaceutical carriers are described in
"Remington's Pharmaceutical Sciences" by E. W. Martin, 18th
Edition, incorporated by reference. Particularly preferred for the
present invention are carriers suitable for immediate-release,
i.e., release of most or all of the active ingredient over a short
period of time, such as 60 minutes or less, and make rapid
absorption of the drug possible.
[0045] The term "pharmaceutically acceptable derivative" as used
herein means any pharmaceutically acceptable salt, solvate or
prodrug, e.g. ester, of a compound of the invention, which upon
administration to the recipient is capable of providing (directly
or indirectly) a compound of the invention, or an active metabolite
or residue thereof Such derivatives are recognizable to those
skilled in the art, without undue experimentation. Nevertheless,
reference is made to the teaching of Burger's Medicinal Chemistry
and Drug Discovery, 5.sup.th Edition, Vol 1: Principles and
Practice, which is incorporated herein by reference to the extent
of teaching such derivatives. Preferred pharmaceutically acceptable
derivatives are salts, solvates, esters, carbamates and phosphate
esters. Particularly preferred pharmaceutically acceptable
derivatives are salts, solvates and esters. Most preferred
pharmaceutically acceptable derivatives are salts and esters.
[0046] The compounds of the present invention may be in the form of
and/or may be administered as a pharmaceutically acceptable salt.
For a review on suitable salts see Berge et al., J. Pharm. Sci.,
1977, 66, 1-19, incorporated by reference.
[0047] Typically, a pharmaceutical acceptable salt may be readily
prepared by using a desired acid or base as appropriate. The salt
may precipitate from solution and be collected by filtration or may
be recovered by evaporation of the solvent. For example, an aqueous
solution of an acid such as hydrochloric acid may be added to an
aqueous suspension of a compound of formula (I) and the resulting
mixture evaporated to dryness (lyophilised) to obtain the acid
addition salt as a solid. Alternatively, a compound of formula (I)
may be dissolved in a suitable solvent, for example an alcohol such
as isopropanol, and the acid may be added in the same solvent or
another suitable solvent. The resulting acid addition salt may then
be precipitated directly, or by addition of a less polar solvent
such as diisopropyl ether or hexane, and isolated by
filtration.
[0048] Suitable addition salts are formed from inorganic or organic
acids which form non-toxic salts and examples are hydrochloride,
hydrobromide, hydroiodide, sulphate, bisulphate, nitrate,
phosphate, hydrogen phosphate, acetate, trifluoroacetate, maleate,
malate, fumarate, lactate, tartrate, citrate, formate, gluconate,
succinate, pyruvate, oxalate, oxaloacetate, trifluoroacetate,
saccharate, benzoate, alkyl or aryl sulfonates (e.g.
methanesulfonate, ethanesulfonate, benzenesulfonate or
p-toluenesulfonate) and isothionate. Representative examples
include trifluoroacetate and formate salts, for example the bis- or
tris-trifluoroacetate salts and the mono or diformate salts, in
particular the bis- or tris-trifluoroacetate salt and the
monoformate salt.
[0049] Pharmaceutically acceptable base salts include ammonium
salts, alkali metal salts such as those of sodium and potassium,
alkaline earth metal salts such as those of calcium and magnesium
and salts with organic bases, including salts of primary, secondary
and tertiary amines, such as isopropylamine, diethylamine,
ethanolamine, trimethylamine, dicyclohexyl amine and
N-methyl-D-glucamine.
[0050] Compounds of the invention may have both a basic and an
acidic center and may therefore be in the form of zwitterions.
[0051] Those skilled in the art of organic chemistry will
appreciate that many organic compounds can form complexes with
solvents in which they are reacted or from which they are
precipitated or crystallized. These complexes are known as
"solvates". For example, a complex with water is known as a
"hydrate". Solvates of the compound of the invention are within the
scope of the invention. The salts of the compound of formula (I)
may form solvates (e.g. hydrates) and the invention also includes
all such solvates.
[0052] The term "prodrug" as used herein means a compound which is
converted within the body, e.g. by hydrolysis in the blood, into
its active form that has medical effects. Pharmaceutically
acceptable prodrugs are described in T. Higuchi and V. Stella,
"Prodrugs as Novel Delivery Systems", Vol. 14 of the A.C.S.
Symposium Series, Edward B. Roche, ed., "Bioreversible Carriers in
Drug Design", American Pharmaceutical Association and Pergamon
Press, 1987, and in D. Fleisher, S. Ramon and H. Barbra "Improved
oral drug delivery: solubility limitations overcome by the use of
prodrugs", Advanced Drug Delivery Reviews (1996) 19(2) 115-130,
each of which are incorporated herein by reference.
[0053] Prodrugs are any covalently bonded carriers that release a
compound of structure (I) in vivo when such prodrug is administered
to a patient. Prodrugs are generally prepared by modifying
functional groups in a way such that the modification is cleaved,
either by routine manipulation or in vivo, yielding the parent
compound. Prodrugs include, for example, compounds of this
invention wherein hydroxy, amine or sulfhydryl groups are bonded to
any group that, when administered to a patient, cleaves to form the
hydroxy, amine or sulfhydryl groups. Thus, representative examples
of prodrugs include (but are not limited to) acetate, formate and
benzoate derivatives of one or more of alcohol, sulfhydryl and
amine functional groups of the compounds of structure (I). Further,
in the case of a carboxylic acid (--COOH) group, esters may be
employed, such as methyl esters, ethyl esters, and the like. Esters
may be active in their own right and/or be hydrolysable under in
vivo conditions in the human body. Suitable pharmaceutically
acceptable in vivo hydrolysable ester groups include those which
break down readily in the human body to leave the parent acid or
its salt.
[0054] References hereinafter to a compound according to the
invention include both compounds of formula (I) and their
pharmaceutically acceptable derivatives.
[0055] With regard to stereoisomers, the compounds of structure (I)
have more than one asymmetric carbon atom. In the general formula
(I) as drawn, the solid wedge shaped bond indicates that the bond
is above the plane of the paper. The broken bond indicates that the
bond is below the plane of the paper.
[0056] It will be appreciated that the substituents on the
macrolide may also have one or more asymmetric carbon atoms. Thus,
the compounds of structure (I) may occur as individual enantiomers
or diastereomers. All such isomeric forms are included within the
present invention, including mixtures thereof.
[0057] Where a compound of the invention contains an alkenyl group,
cis (Z) and trans (E) isomerism may also occur. The present
invention includes the individual stereoisomers of the compound of
the invention and, where appropriate, the individual tautomeric
forms thereof, together with mixtures thereof.
[0058] Separation of diastereoisomers or cis and trans isomers may
be achieved by conventional techniques, e.g. by fractional
crystallisation, chromatography or H.P.L.C. An enantio- and
diastereopure or enriched agent may also be prepared from a
corresponding optically pure intermediate or by resolution, such as
H.P.L.C., of the corresponding racemate using a suitable chiral
support or by fractional crystallisation of the diastereoisomeric
salts formed by reaction of the corresponding racemate with a
suitable optically active acid or base, as appropriate.
[0059] The compounds of Formula (I) may be in crystalline or
amorphous form. Furthermore, some of the crystalline forms of the
compounds of Formula (I) may exist as polymorphs, which are
included in the present invention.
[0060] Compounds wherein R.sup.2 represents a hydroxyl protecting
group are in general intermediates for the preparation of other
compounds of Formula (I).
[0061] When the group OR.sup.2 is a protected hydroxyl group this
is conveniently an ether or an acyloxy group. Examples of
particularly suitable ether groups include those in which R.sup.2
is a trialkylsilyl (i.e. trimethylsilyl). When the group OR.sup.2
represents an acyloxy group, then examples of suitable groups
R.sup.2 include acetyl, benzoyl or benzyloxycarbonyl.
[0062] The term "alkyl" as used herein as a group or a part of a
group refers to a straight or branched hydrocarbon chain containing
the specified number of carbon atoms. For example, C.sub.1-6 alkyl
means a straight or branched alkyl chain containing from 1 to 6
carbon atoms; examples of such group include methyl, ethyl, propyl,
isopropyl, n-butyl, isobutyl, tert-butyl, pentyl, 3-methyl-butyl,
hexyl and 2,3-dimethylbutyl and like.
[0063] When an alkyl chain is interrupted by 1-3 of --O--, --S-- or
--N(R.sup.7)-- a methylene spacer can be present adjacent to an
interrupting moiety. Thus, this would include, for example,
--CH.sub.2--O-- and --O--CH.sub.2--. When two or three of these
interrupting moieties are present, they are separated from one
another by at least one methylene spacer.
[0064] The term "alkenyl" as used herein as a group or a part of a
group refers to a straight or branched hydrocarbon chain containing
the specified number of carbon atoms and containing at least one
double bond. For example, the term "C.sub.2-6 alkenyl" means a
straight or branched alkenyl containing at least 2, and at most 6,
carbon atoms and containing at least one double bond. Examples of
"alkenyl" as used herein include, but are not limited to, ethenyl,
2-propenyl, 3-butenyl, 2-butenyl, 2-pentenyl, 3-pentenyl,
3-methyl-2-butenyl, 3-methylbut-2-enyl, 3-hexenyl and
1,1-dimethylbut-2-enyl. Interrupting moieties as above can be
present within alkenyl chains. It will be appreciated that in
groups of the form --O--C.sub.2-6 alkenyl, the double bond is
preferably not adjacent to the oxygen.
[0065] The term "C.sub.1-5 alkanoyl" refers to acyl group such as
formyl, acetyl, propanoyl or butanoyl.
[0066] The term "halogen" refers to a fluorine, chlorine, bromine
or iodine atom.
[0067] The term "C.sub.3-7 cycloalkyl" group as used herein refers
to a non-aromatic monocyclic hydrocarbon ring of 3 to 7 carbon
atoms such as, for example, cyclopropyl, cyclobutyl, cyclopentyl,
cyclohexyl or cycloheptyl.
[0068] The term "alkoxy" as used herein refers to a straight or
branched chain alkoxy group containing the specified number of
carbon atoms. For example, C.sub.1-6 alkoxy means a straight or
branched alkoxy containing at least 1, and at most 6, carbon atoms.
Examples of "alkoxy" as used herein include, but are not limited
to, methoxy, ethoxy, propoxy, prop-2-oxy, butoxy, but-2-oxy,
2-methylprop-1-oxy, 2-methylprop-2-oxy, pentoxy and hexyloxy.
[0069] The term "hydroxyalkyl" as used herein as a group refers to
a straight or branched hydrocarbon chain containing the specified
number of carbon atoms, which is substituted by 1-3 hydroxyl
groups. For example, C.sub.1-4 hydroxyalkyl means a straight or
branched alkyl chain containing from 1 to 4 carbon atoms and at
least one hydroxyl group; examples of such groups include
hydroxymethyl, hydroxyethyl, hydroxypropyl, hydroxyisopropyl,
hydroxybutyl and like.
[0070] The term "heterocyclic" as used herein, refers to a
non-aromatic, saturated or unsaturated monocycle, containing two-
to six carbon atoms and at least one heteroatom selected from
oxygen, nitrogen and sulfur. Preferably, the heterocyclyl ring has
five to seven ring atoms. Examples of heterocyclyl groups include,
but are not limited to, pyrrolidinyl, tetrahydrofuranyl,
tetrahydrothiophenyl, imidazolidinyl, pyrazolidinyl, piperidinyl,
piperazinyl, hoinopiperazinyl, hexamethyleneiminyl, morpholinyl,
tetrahydropyranyl and thiomorpholinyl.
[0071] The term "leaving group" refers to a chemical group which is
capable of being displaced by a nucleophile. Examples of such
groups include but are not limited to halogen, mesylate, tosylate
and ester groups.
[0072] In some preferred embodiments, this invention relates to the
compounds of Formula (I) and to pharmaceutically acceptable
derivatives thereof wherein A represent --NHC(O)-- or --C(O)NH--.
Within this subset, all other variables are as originally
defined.
[0073] Another preferred aspect of the invention relates to the
compounds of Formula (I) and to pharmaceutically acceptable
derivatives thereof wherein A represent --N(R.sup.7)CH.sub.2-- or
--CH.sub.2N(R.sup.7)--. Within this subset, all other variables are
as originally defined.
[0074] Yet another preferred aspect of the invention.degree.
relates to the compounds of Formula (I) and to pharmaceutically
acceptable derivatives thereof wherein A represent --C(O)--,
--CH(OH)--, or --C(.dbd.NOR.sup.7)--. Within this subset, all other
variables are as originally defined.
[0075] Representative examples of R.sup.3 include hydrogen,
unsubstituted C.sub.1-4 alkyl, for example methyl, substituted
C.sub.1-4 alkyl, for example C.sub.1-4 aminoalkyl or C.sub.1-4
cyanoalkyl and C.sub.1-5 alkanoyl, for example acetyl.
[0076] A preferred aspect of the invention relates to the compounds
of Formula (I) and to pharmaceutically acceptable derivatives
thereof wherein R.sup.4 is hydrogen or methyl.
[0077] In one embodiment, R.sup.5 is hydroxy or methoxy and R.sup.6
is hydroxy. Alternatively, R.sup.5 and R.sup.6 taken together with
the intervening atoms form a cyclic group having the following
structure:
##STR00004##
wherein Y is a bivalent radical selected from --O-- and
--N(R.sup.7)--.
[0078] A "pharmaceutically acceptable excipient" means an excipient
that is useful in preparing a pharmaceutical composition that is
generally safe, non-toxic and neither biologically nor otherwise
undesirable, and includes an excipient that is acceptable for
veterinary use as well as human pharmaceutical use. A
"pharmaceutically acceptable excipient" as used in the present
application includes both one and more than one such excipient.
[0079] "Treating" or "treatment" of a state, disorder or condition
includes:
[0080] (1) preventing or delaying the appearance of clinical
symptoms of the state, disorder or condition developing in a mammal
that may be afflicted with or predisposed to the state, disorder or
condition but does not yet experience or display clinical or
subclinical symptoms of the state, disorder or condition,
[0081] (2) inhibiting the state, disorder or condition, i.e.,
arresting or reducing the development of the disease or at least
one clinical or subclinical symptom thereof, or
[0082] (3) relieving the disease, i.e., causing regression of the
state, disorder or condition or at least one of its clinical or
subclinical symptoms.
[0083] The benefit to a subject to be treated is either
statistically significant or at least perceptible to the patient or
to the physician.
[0084] A "therapeutically effective amount" means the amount of a
compound that, when administered to a mammal for treating a state,
disorder or condition, is sufficient to effect such treatment. The
"therapeutically effective amount" will vary depending on the
compound, the disease and its severity and the age, weight,
physical condition and responsiveness of the mammal to be
treated.
[0085] The four classic symptoms of acute inflammation are redness,
elevated temperature, swelling, and pain in the affected area, and
impairment or loss of function of the affected organ.
[0086] Symptoms and signs of inflammation associated with specific
conditions include: [0087] rheumatoid arthritis-pain, swelling,
warmth and tenderness of the involved joints; generalized and
morning stiffness; [0088] insulin-dependent diabetes
mellitus-insulitis; this condition can lead to a variety of
complications with an inflammatory component, including:
retinopathy, neuropathy, nephropathy; coronary artery disease,
peripheral vascular disease, and cerebrovascular disease; [0089]
autoimmune thyroiditis-weakness, constipation, shortness of breath,
puffiness of the face, hands and feet, peripheral edema,
bradycardia; [0090] multiple sclerosis-spasticity, blurry vision,
vertigo, limb weakness, paresthesias; [0091]
uveoretinitis-decreased night vision, loss of peripheral vision;
[0092] lupus erythematosus-joint pain, rash, photosensitivity,
fever, muscle pain, puffiness of the hands and feet, abnormal
urinalysis (hematuria, cylinduria, proteinuria),
glomerulonephritis, cognitive dysfunction, vessel thrombosis,
pericarditis; [0093] scleroderma- Raynaud's disease; swelling of
the hands, arms, legs and face; skin thickening; pain, swelling and
stiffness of the fingers and knees, gastrointestinal dysfunction,
restrictive lung disease; pericarditis; renal failure; [0094] other
arthritic conditions having an inflammatory component such as
rheumatoid spondylitis, osteoarthritis, septic arthritis and
polyarthritis-fever, pain, swelling, tenderness; [0095] other
inflammatory brain disorders, such as meningitis, Alzheimer's
disease, AIDS dementia encephalitis-photophobia, cognitive
dysfunction, memory loss; [0096] other inflammatory eye
inflammations, such as retinitis-decreased visual acuity; [0097]
inflammatory skin disorders, such as, eczema, other dermatites
(e.g., atopic, contact), psoriasis, burns induced by UV radiation
(sun rays and similar UV sources)-erythema, pain, scaling,
swelling, tenderness; [0098] inflammatory bowel disease, such as
Crohn's disease, ulcerative colitis-pain, diarrhea, constipation,
rectal bleeding, fever, arthritis; [0099] asthma-shortness of
breath, wheezing; [0100] other allergy disorders, such as allergic
rhinitis-sneezing, itching, runny nose [0101] conditions associated
with acute trauma such as cerebral injury following stroke-sensory
loss, motor loss, cognitive loss; [0102] heart tissue injury due to
myocardial ischemia-pain, shortness of breath; [0103] lung injury
such as that which occurs in adult respiratory distress
syndrome-shortness of breath, hyperventilation, decreased
oxygenation, pulmonary infiltrates; [0104] inflammation
accompanying infection, such as sepsis, septic shock,
osteomyelitis, toxic shock syndrome-fever, respiratory failure,
tachycardia, hypotension, leukocytosis; [0105] other inflammatory
conditions associated with particular organs or tissues, such as
nephritis (e.g., glomerulonephritis)-oliguria, abnormal urinalysis;
[0106] inflamed appendix-fever, pain, tenderness, leukocytosis;
[0107] gout-pain, tenderness, swelling and erythema of the involved
joint, elevated serum and/or urinary uric acid; [0108] inflamed
gall bladder-abdominal pain and tenderness, fever, nausea,
leukocytosis; [0109] chronic obstructive pulmonary disorder
(COPD)-shortness of breath, wheezing; [0110] congestive heart
failure-shortness of breath, rales, peripheral edema, chronic
sinusitis, nasal polyps; cystic fibrosis; diffuse panbronchiolitis;
bronchiectasis; bronchiolitis obliterans; [0111] Type II
diabetes-end organ complications including cardiovascular, ocular,
renal, peripheral vascular disease and coronary artery disease;
[0112] lung fibrosis (i.e., pulmonary fibrosis)-hyperventilation,
shortness of breath, decreased oxygenation; [0113] vascular
disease, such as atherosclerosis and restenosis-pain, loss of
sensation, diminished pulses, loss of function; and [0114]
alloimmunity leading to transplant rejection-pain, tenderness,
fever.
[0115] Symptoms associated with COPD have been outlined supra.
[0116] Subclinical symptoms include without limitation diagnostic
markers for inflammation the appearance of which may precede the
manifestation of clinical symptoms. One class of subclinical
symptoms is immunological symptoms, such as the invasion or
accumulation in an organ or tissue of proinflammatory lymphoid
cells or the presence locally or peripherally of activated
pro-inflammatory lymphoid cells recognizing a pathogen or an
antigen specific to the organ or tissue and secreting or inducing
pro-inflammatory cytokines. Activation of lymphoid cells can be
measured by techniques known in the art.
[0117] "Delivering" a therapeutically effective amount of an active
ingredient to a particular location within a host means causing a
therapeutically effective blood concentration of the active
ingredient at the particular location. This can be accomplished,
e.g., by local or by systemic administration of the active
ingredient to the host.
[0118] The term host or subject in need thereof as used herein
refers to a mammal preferably a human.
[0119] Preferred compounds of the invention are the compounds of
examples 1-98 and pharmaceutically acceptable derivatives
thereof.
[0120] Methods of Preparation:
[0121] Compounds of Formula (I) and pharmaceutically acceptable
derivatives thereof may be prepared by the general methods outlined
hereinafter, said methods constituting a further aspect of the
invention. In the following description, the groups R.sup.1 to
R.sup.9, A and n, have the meaning defined for the compounds of
Formula (I) unless otherwise stated.
[0122] It will be obvious to a person skilled in the art that, in
order to avoid interference with any functional groups other than
those where structural modifications are to be made, appropriate
protection and priority in the synthetic route should be
chosen.
[0123] The synthesis of the target compound is completed by
removing any protecting groups, which are present in the
penultimate intermediate using standard techniques, which are well
known to those skilled in the art. The deprotected final product is
then purified, as necessary, using standard techniques such as
silica gel chromatography, HPLC on silica gel, and the like or by
recrystallization.
[0124] The group --NR.sup.8aR.sup.9a in the following synthetic
paths is --NR.sup.8R.sup.9 as defined for formula (I) or a group
convertible to --NR.sup.8R.sup.9. Conversion of a group
--NR.sup.8aR.sup.9a to a --NR.sup.8R.sup.9 group typically arises
if a protecting group is needed during the reactions described
below. A comprehensive discussion of the ways in which such groups
may be protected and methods for cleaving the resulting protected
derivatives is given by for example T. W. Greene and P. G. M Wuts
in Protective Groups in Organic Synthesis 2.sup.nd ed., John Wiley
& Son, Inc 1991, and by P. J. Kocienski in Protecting Groups,
Georg Thieme Verlag 1994. Examples of suitable amino protecting
groups include acyl type protecting groups (e.g. formyl,
trifluoroacetyl and acetyl), aromatic urethane type protecting
groups (e.g. benzyloxycarbonyl (Cbz) and substituted Cbz, and
9-fluorenylmethoxycarbonyl (Fmoc)), aliphatic urethane protecting
groups (e.g. t-butyloxycarbonyl (Boc), isopropyloxycarbonyl and
cyclohexyloxycarbonyl) and alkyl type protecting groups (e.g.
benzyl, trityl and chlorotrityl). Examples of suitable oxygen
protecting groups may include for example alkyl silyl groups, such
as trimethylsilyl or tert-butyldimethylsilyl; alkyl ethers such as
tetrahydropyranyl or tert-butyl; or esters such as acetate. Hydroxy
groups may be protected by reaction of for example acetic
anhydride, benzoic anhydride or a trialkylsilyl chloride in an
aprotic solvent. Examples of aprotic solvents are dichloromethane,
N,N-dimethylformamide, dimethylsulfoxide, tetrahydrofuran and the
like.
[0125] The compounds of formula (I) wherein R.sup.1 is
OC(O)(CH.sub.2).sub.nNR.sup.8R.sup.9 and n is an integer from 1 to
8 may be prepared by reaction of the compounds of formula (II)
wherein R.sup.2 is a hydroxy protecting group with carboxylic acid
or a suitable activated derivative of carboxylic acid (III),
followed where necessary by subsequent removal of the hydroxyl
protecting group R.sup.2 and conversion of the --NR.sup.8aR.sup.9a
group to --NR.sup.8R.sup.9. Similarly, intermediate compounds
related to the compounds of formula (I) wherein R.sup.1 is
OC(O)(CH.sub.2).sub.n NR.sup.8R.sup.9 and R.sup.3 is CH.sub.3 may
be prepared by the same method.
##STR00005##
[0126] Suitable activated derivatives of the carboxylic acid
include the corresponding acyl halide, mixed anhydride or activated
ester such as a thiol ester.
[0127] The reaction is preferably carried out in a suitable aprotic
solvent such as a halohydrocarbon (e.g. dichloromethane) or
N,N-dimethylformamide optionally in the presence of a tertiary base
such as dimethylaminopyridine or triethylamine or in the presence
of inorganic base (e.g. sodium hydroxide) and at a temperature
within the range of 0.degree. to 120.degree. C. The compounds of
formula (II) and (III) may also be reacted in the presence of a
carbodiimide such as dicyclohexylcarbodiimide (DCC).
[0128] In a further embodiment of the invention, compounds of
formula (I) wherein R.sup.1 is OC(O)(CH.sub.2).sub.nNR.sup.8R.sup.9
and n is an integer from 1 to 8 may be prepared by reaction of the
compounds of formula (IV) wherein n is an integer from 1 to 8 and L
is a suitable leaving group, with NR.sup.8aR.sup.8a (V).
##STR00006##
[0129] The reaction is preferably carried out in a solvent such as
a halohydrocarbon (e.g. dichloromethane), an ether (e.g.
tetrahydrofuran or dimethoxyethane), acetonitrile or ethyl acetate
and the like, dimethylsulfoxide, N,N-dimethylformamide or
1-methyl-pyrrolidone and in the presence of a base, followed, if
desired, by removal of the hydroxyl protecting group R.sup.2 and
conversion of the --NR.sup.8aR.sup.9a group to --NR.sup.8R.sup.9
Examples of the bases which may be used include organic bases such
as diisopropylethylamine, triethylamine and
1,8-diazabicyclo[5.4.0]undec-7-ene, and inorganic bases such as
potassium hydroxide, cesium hydroxide, tetraalkylammonium
hydroxide, sodium hydride, potassium hydride and the like. Suitable
leaving groups for this reaction include halide (e.g. chloride,
bromide or iodide) or a sulfonate group (e.g. tosylate,
methanesulfonate, or triflate).
[0130] Compounds of formula (IV) may be prepared by reaction of a
compound of formula (II), wherein R.sup.2 is a hydroxyl protecting
group, with carboxylic acid (VI) or a suitable activated derivative
of the carboxylic acid HOC(O)(CH.sub.2).sub.nL (VI), wherein L is a
suitable leaving group as defined in the previous paragraph.
Suitable activated derivatives of the carboxyl group are those
defined above for carboxylic acid (III). The reaction is carried
out using the conditions described above for the reaction of a
compound of formula (II) with carboxylic acid (III).
[0131] In a preferred embodiment of the invention, compounds of
formula (I) wherein R.sup.1 is OC(O)(CH.sub.2).sub.nNR.sup.8R.sup.9
and n is 2 may be prepared by Michael reaction of the compounds of
formula (VII) wherein R.sup.2 is a hydroxy protecting group with a
compound of formula N.sub.R8aR.sup.9a(V).
##STR00007##
[0132] The reaction is suitably carried out in a solvent such as
dimethylsulfoxide, N,N-dimethylformamide, 1-methyl-pyrrolidone, a
halohydrocarbon (e.g. dichloromethane), an ether (e.g.
tetrahydrofuran or dimethoxyethane), acetonitrile or alcohol (e.g.
methanol or isopropanol) and the like, and in the presence of a
base, followed, if desired, by removal of hydroxyl protecting group
R.sup.2 and conversion of the --NR.sup.8aR.sup.9a group to
--NR.sup.8R.sup.9.
[0133] Compounds of formula (VII) can be prepared according to the
procedure described in international patent application WO
03/042228 incorporated by reference in its entirety, especially
pages 16-18. Thus, reaction of the compounds of formula (II)
wherein R.sup.2 is a hydroxy protecting group with
3-chloropropionyl chloride in the presence of a base such as
triethylamine gave compounds of formula (VII).
[0134] Compounds of formula (I) wherein R.sup.1 is
--O--(CH.sub.2).sub.nNR.sup.8R.sup.9 may be prepared by reaction of
a 4''-aldehyde compound of formula (VIII), wherein n is an integer
from 1 to 8, with a compound of formula NR.sup.8aR.sup.9a (V),
followed where necessary by subsequent removal of the protecting
groups and conversion of the NR.sup.8aR.sup.9a to
NR.sup.8R.sup.9.
##STR00008##
[0135] The reductive amination reaction is preferably carried out
in a solvent such as methanol and DMF. A suitable reducing agent
is, for example, sodium cyanoborohydride.
[0136] Compounds of formula (VIII) where n is 1 or 2 may be
prepared from suitably protected compounds of formula (IX) by
hydroboration with 9-BBN, or other suitable boranes, followed by
treatment with peroxide and then oxidation (n=2), or by osmium
tetroxide/peridoate cleavage (n=1).
[0137] Compounds of formula (IX) can be formed by
palladium-catalyzed allylation (Red. Tray. Chim. Pays-Bas 102,
501-505, 1983) of the 4'' the hydroxy compounds of formula (II)
that are suitably protected, for example by cyclic protection
between the 9 and 11 positions when A is --C(OH)-- (J. Antibiot.,
42, 293, 1989.).
##STR00009##
[0138] In a further embodiment of the invention, compounds of
formula (I) wherein R.sup.1 is --O--(CH.sub.2).sub.nNR.sup.8R.sup.9
may be prepared by reaction of compounds of foimula (X) with a
##STR00010##
compound of formula NR.sup.8aR.sup.9a (V), wherein n is an integer
from 2 to 8 and L is suitable leaving group. The reaction is
preferably carried out in a solvent such as a halohydrocarbon (e.g.
dichloromethane), an ether (e.g. tetrahydrofuran or
dimethoxyethane), acetonitrile or ethyl acetate and the like,
dimethylsulfoxide, N,N-dimethylformamide or 1-methyl-pyrrolidone
and in the presence of a base, followed, if desired, by removal of
protecting group R.sup.2 and conversion of the NR.sup.8aR.sup.9a
group to NR.sup.8R.sup.9. Examples of the bases which may be used
include organic bases such as diisopropylethylamine, triethylamine
and 1,8-diazabicyclo[5.4.0]undec-7-ene (DBU), and inorganic bases
such as potassium hydroxide, cesium hydroxide, tetraalkylammonium
hydroxide, sodium hydride, potassium hydride and the like. Suitable
leaving groups for this reaction are halide (e.g. chloride, bromide
or iodide).
[0139] In a further embodiment of the invention, compounds of
formula (I) wherein R.sup.1 is
--O--(CH.sub.2).sub.nNR.sup.8R.sup.9, n is 3, R.sup.8 and R.sup.9
are the same and have the meaning as defined above may be prepared
by reductive alkylation of 4''-amine of formula (XI) wherein Z is
CH.sub.2NH.sub.2 with a compound of formula HC(O)R.sup.8 (XII).
##STR00011##
[0140] Compound of formula (XI) wherein Z is CH.sub.2NH.sub.2 may
be prepared by reaction of suitable protected compound of formula
(II) with acrylonitrile in solvent such as DMSO, THF, t-BuOH in the
presence of a base such as NaH giving compound of formula (XI)
wherein Z is cyano group, followed by catalytic reduction of cyano
group.
[0141] Compounds of formula (I) may be prepared by reaction of a
suitable activated compound of formula (XII) wherein R.sup.2 is
optionally a hydroxy protecting group and R.sup.10 is an activating
group such as imidazolyl or halogen, with a suitable protected
derivative of the amine of formula (XIIIa) or (XIIIb), followed
where necessary by subsequent removal of the hydroxyl protecting
group R.sup.2 and conversion of the NR.sup.8aR.sup.9a group to
NR.sup.8R.sup.9.
##STR00012##
[0142] The reaction is preferably carried out in a suitable aprotic
solvent such as N,N-dimethylformamide in the presence of an organic
base such as 1,8-diazabiyclo[5.4.0]undec-7-ene (DBU).
[0143] Compounds of formula (II) wherein A is --C(.dbd.NOR.sup.7)
and R.sup.7 is hydrogen are known compounds or they may be prepared
by conventional techniques, for example according to U.S. Pat. No.
3,478,014 or Journal of Antibiotics, 44, 313, 1991. Compounds
wherein R.sup.7 is other than a hydrogen atom may be prepared by
alkylation of the oxime, for instance according to EP 1 167 375.
All are incorporated by reference.
[0144] Compounds of formula (II) wherein A is --CH(OH)-- are known
compounds or they may be prepared by conventional techniques, for
example by treatment of C-9 keto group with reducing agents, for
instance hydrides (sodium borohydride or sodium cyanoborohydride
(JACS 79, 6062, 1957, Journal of Antibiotics 43.1334, 1990,
incorporated by reference).
[0145] Compounds of formula (II) wherein A is --NHC(O)-- or
--C(O)NH-- and R.sup.4 is C.sub.1-4 alkyl or C.sub.2-6 alkenyl are
known compounds or they may be prepared from corresponding
6-O-alkyl or alkenylerythromycin A oxime by Beckman rearrangement
according to the procedure described in WO 9951616, incorporated by
reference.
[0146] Compounds of formula (II) wherein A is --NR.sup.7CH.sub.2--
or --CH.sub.2N(R.sup.7)-- are known compounds or they may be
prepared by analogous methods to those known in the art. Thus, they
can be prepared according to the procedures described in U.S. Pat.
No. 4,328,334, BE 892357, U.S. Pat. No. 4,464,527, Bioorg. Med.
Chem. Lett., 3, 1287, 1993, all incorporated by reference.
[0147] Compounds of formula (II) wherein R.sup.3 is C.sub.2-4 alkyl
or C.sub.1-5 alkanoyl are prepared by mono-demethylation of the
3'-NMe.sub.2 group with benzylchloroformate, followed by
elimination benzyloxycarbonyl groups at position 2' and 3' as
described in U.S. Pat. No. 5,250,518. An alternative method for
demethylation of the 3'-NMe.sub.2 group may be performed by
treatment with sodium acetate and iodine in the presence of an
organic solvent, as described in U.S. Pat. No. 3,725,385 and WO
2004/013153. The subsequent alkylation or acylation of the
secondary amine thus obtained are carried out in accordance with
conventional synthetic techniques. All documents cited in this
paragraph are incorporated by reference.
[0148] Compounds of formula (II) wherein R.sup.5 and R.sup.6 taken
together with the intervening atoms form a cyclic group having the
following structure:
##STR00013##
wherein Y is a bivalent radical selected from --CH.sub.2--,
--CH(CN)--, --O--, --N(R.sup.7)-- and --CH(SR.sup.7)--; may be
prepared by analogous methods to those skilled in the art. Thus,
they can be prepared according to the procedure described in WO
2004/039822, herein incorporated by reference, and by references
cited therein.
[0149] A further aspect of the present invention relates to methods
for using the compounds of Formula I as anti-inflammatory, and
immunomodulating agents which can be administered in different
ways, depending on the inflammation site, e.g. percutaneously,
orally, buccally, rectally, parenterally or by inhalation when
application within the respiratory tract is intended.
[0150] The corresponding preparations of the compounds of the
present invention can be used in the prophylaxis as well as in the
therapeutic treatment (prevention, delay, inhibition or relief) of
several disorders (diseases and other pathological inflammatory
conditions) caused by or associated with an abnormal or undesirable
(excessive, nonregulated, or dysregulated) inflammatory immune
response involving the production of inflammatory cytokines or
other inflammation mediators, including without limitation
TNF-.alpha., IL-1, IL-2, IL-5, IL6, and IL-8.
[0151] These disorders include autoimmune diseases such as
rheumatoid arthritis, insulin-dependent diabetes mellitus,
autoimmune thyroiditis, multiple sclerosis, uveoretinitis, lupus
erythematosus, scleroderma; psoriasis, acne; other arthritic
conditions having an inflammatory component such as rheumatoid
spondylitis, osteoarthritis, osteomyelitis; septic arthritis and
polyarthritis; other inflammatory brain disorders, such as
meningitis, Alzheimer's disease, AIDS dementia encephalitis, other
inflammatory eye inflammations, such as retinitis; inflammatory
skin disorders, such as, eczema, other dermatites (e.g., atopic,
contact), psoriasis, burns induced by UV radiation (sun rays and
similar UV sources); inflammatory bowel disease, such as Crohn's
disease, ulcerative colitis; COPD; cystic fibrosis; bronchiectasis;
asthma; other allergy disorders, such as allergic rhinitis; chronic
sinusitis; conditions associated with acute trauma such as cerebral
injury following stroke, heart tissue injury due to myocardial
ischemia, lung injury such as that which occurs in adult
respiratory distress syndrome; inflammation accompanying infection,
such as sepsis, septic shock, toxic shock syndrome, other
inflammatory conditions associated with particular organs or
tissues, such as nephritis (e.g., glomerulonephritis), inflamed
appendix, gout, inflamed gall bladder, congestive heart failure,
Type II diabetes, lung fibrosis, bronchiolitis obliterans; diffuse
panbronchiolitis; vascular disease, such as atherosclerosis and
restenosis; and alloimmunity leading to transplant rejection. The
compounds can also be administered by inhalation when application
within the respiratory tract is intended. A further object of the
present invention relates to the preparation of various
pharmaceutical forms of the compounds to achieve the optimal
bioavailability of the active compound of Formula I.
[0152] Pharmaceutical Compositions
[0153] Further, the present invention relates to pharmaceutical
compositions containing an effective dose of compounds of the
present invention as well as pharmaceutically acceptable
excipients, such as carriers or diluents.
[0154] While it is possible that, for use in the methods of the
invention, a compound of formula I may be administered as the bulk
substance, it is preferable to present the active ingredient in a
pharmaceutical formulation, e.g., wherein the agent is in admixture
with a pharmaceutically acceptable carrier selected with regard to
the intended route of administration and standard pharmaceutical
practice.
[0155] The corresponding preparations of the compounds of the
present invention can be used in the prophylaxis (including without
limitation the prevention, delay or inhibition of recurrence of one
or more of the clinical or subclinical symptoms discussed and
defined in connection with the definitions of "treatment" above, as
well as in the therapeutic treatment of several diseases and
pathological inflammatory conditions including: chronic obstructive
pulmonary disorder (COPD), asthma, inflammatory nasal diseases such
as allergic rhinitis, nasal polyps, intestinal diseases such as
Crohn's disease, colitis, intestinal inflammation, ulcerative
colitis, dermatological inflammations such as eczema, psoriasis,
allergic dermatitis, neurodermatitis, pruritis, conjunctivitis and
rheumatoid arthritis.
[0156] The term "carrier" refers to a diluent, excipient, and/or
vehicle with which an active compound is administered. The
pharmaceutical compositions of the invention may contain
combinations of more than one carrier. Such pharmaceutical carriers
can be sterile liquids, such as water, saline solutions, aqueous
dextrose solutions, aqueous glycerol solutions, and oils, including
those of petroleum, animal, vegetable or synthetic origin, such as
peanut oil, soybean oil, mineral oil, sesame oil and the like.
Water or aqueous solution saline solutions and aqueous dextrose and
glycerol solutions are preferably employed as carriers,
particularly for injectable solutions. Suitable pharmaceutical
carriers are described in "Remington's Pharmaceutical Sciences" by
E. W. Martin, 18th Edition. The choice of pharmaceutical carrier
can be selected with regard to the intended route of administration
and standard pharmaceutical practice. The pharmaceutical
compositions may comprise as, in addition to, the carrier any
suitable binder(s), lubricant(s), suspending agent(s), coating
agent(s), and/or solubilizing agent(s).
[0157] A "pharmaceutically acceptable excipient" means an excipient
that is useful in preparing a pharmaceutical composition that is
generally safe, non-toxic and neither biologically nor otherwise
undesirable, and includes an excipient that is acceptable for
veterinary use as well as human pharmaceutical use. A
"pharmaceutically acceptable excipient" as used in the present
application includes both one and more than one such excipient.
[0158] It will be appreciated that pharmaceutical compositions for
use in accordance with the present invention may be in the form of
oral, parenteral, transdermal, inhalation, sublingual, topical,
implant, nasal, or enterally administered (or other mucosally
administered) suspensions, capsules or tablets, which may be
formulated in conventional manner using one or more
pharmaceutically acceptable carriers or excipients.
[0159] There may be different composition/formulation requirements
depending on the different delivery systems. It is to be understood
that not all of the compounds need to be administered by the same
route. Likewise, if the composition comprises more than one active
component, then those components may be administered by the same or
different routes. By way of example, the pharmaceutical composition
of the present invention may be formulated to be delivered using a
mini-pump or by a mucosal route, for example, as a nasal spray or
aerosol for inhalation or ingestible solution, or parenterally in
which the composition is formulated by an injectable form, for
delivery, by, for example, an intravenous, intramuscular or
subcutaneous route. Alternatively, the formulation may be designed
to be delivered by multiple routes.
[0160] The present invention further relates to pharmaceutical
formulations containing a therapeutically effective quantity of a
compound of formula I or one of its salts mixed with a
pharmaceutically acceptable vehicle. The pharmaceutical
formulations of the present invention can be liquids that are
suitable for oral, mucosal and/or parenteral administration, for
example, drops, syrups, solutions, injectable solutions that are
ready for use or are prepared by the dilution of a freeze-dried
product but are preferably solid or semisolid as tablets, capsules,
granules, powders, pellets, pessaries, suppositories, creams,
salves, gels, ointments; or solutions, suspensions, emulsions, or
other forms suitable for administration by the transdennal route or
by inhalation.
[0161] The compounds of the invention can be administered for
immediate-, delayed-, modified-, sustained-, pulsed-or
controlled-release applications.
[0162] The compound can also be incorporated into a formulation for
treating inflammation localized in an organ or tissue, e.g.,
Crohn's disease, where it can be administered orally or rectally.
Formulations for oral administration can incorporate excipients
enabling bioavailability of the compound at the site of
inflammation. This can be achieved by different combinations of
enteric and delayed release formulations. The compound of Formula I
can also be used in the treatment of Crohn's disease and intestinal
inflammation disease if the compound is applied in the form of a
clyster, for which a suitable formulation can be used, as is well
known in the field.
[0163] In some embodiments, the oral compositions are slow, delayed
or positioned release (e.g., enteric especially colonic release)
tablets or capsules. This release profile can be achieved without
limitation by use of a coating resistant to conditions within the
stomach but releasing the contents in the colon or other portion of
the GI tract wherein a lesion or inflammation site has been
identified. Or a delayed release can be achieved by a coating that
is simply slow to disintegrate. Or the two (delayed and positioned
release) profiles can be combined in a single formulation by choice
of one or more appropriate coatings and other excipients. Such
formulations constitute a further feature of the present
invention.
[0164] Formulations for oral administration can be so designed to
enable bioavailability of the compound at the site of inflammation
in the intestines. This can be achieved by different combinations
of delayed release formulations. The compound of Formula I can also
be used in the treatment of Crohn's disease and intestinal
inflammation disease if the compound is applied in the form of an
enema, for which a suitable formulation can be used.
[0165] Suitable compositions for delayed or positioned release
and/or enteric coated oral formulations include tablet formulations
film coated with materials that are water resistant, pH sensitive,
digested or emulsified by intestinal juices or sloughed off at a
slow but regular rate when moistened. Suitable coating materials
include, but are not limited to, hydroxypropyl methylcellulose,
ethyl cellulose, cellulose acetate phthalate, polyvinyl acetate
phthalate, hydroxypropyl methylcellulose phthalate, polymers of
metacrylic acid and its esters, and combinations thereof.
Plasticizers such as, but not limited to polyethylene glycol,
dibutylphthalate, triacetin and castor oil may be used. A pigment
may also be used to color the film. Suppositories are be prepared
by using carriers like cocoa butter, suppository bases such as
Suppocire C, and Suppocire NA50 (supplied by Gattefosse Deutschland
GmbH, D-Weil am Rhein, Germany) and other Suppocire type excipients
obtained by interesterification of hydrogenated palm oil and palm
kernel oil (C8-C18 triglycerides), esterification of glycerol and
specific fatty acids, or polyglycosylated glycerides, and whitepsol
(hydrogenated plant oils derivatives with additives). Enemas are
formulated by using the appropriate active compound according to
the present invention and solvents or excipients for suspensions.
Suspensions are produced by using micronized compounds, and
appropriate vehicle containing suspension stabilizing agents,
thickeners and emulsifiers like carboxymethylcellulose and salts
thereof, polyacrylic acid and salts thereof, carboxyvinyl polymers
and salts thereof, alginic acid and salts thereof, propylene glycol
alginate, chitosan, hydroxypropylcellulose,
hydroxypropylmethylcellulose, hydroxyethylcellulose,
ethylcellulose, methylcellulose, polyvinyl alcohol, polyvinyl
pyrolidone, N-vinylacetamide polymer, polyvinyl methacrylate,
polyethylene glycol, pluronic, gelatin, methyl vinyl ether-maleic
anhydride copolymer, soluble starch, pullulan and a copolymer of
methyl acrylate and 2-ethylhexyl acrylate lecithin, lecithin
derivatives, propylene glycol fatty acid esters, glycerin fatty
acid esters, sorbitan fatty acid esters, polyoxyethylene sorbitan
fatty acid esters, polyethylene glycol fatty acid esters,
polyoxyethylene hydrated caster oil, polyoxyethylene alkyl ethers,
and pluronic and appropriate buffer system in pH range of 6.5 to 8.
The use of preservatives, masking agents is suitable. The average
diameter of micronized particles can be between 1 and 20
micrometers, or can be less than 1 micrometer. Compounds can also
be incorporated in the formulation by using their water-soluble
salt forms.
[0166] Alternatively, materials may be incorporated into the matrix
of the tablet e.g. hydroxypropyl methylcellulose, ethyl cellulose
or polymers of acrylic and metacrylic acid esters. These latter
materials may also be applied to tablets by compression
coating.
[0167] Pharmaceutical compositions can be prepared by mixing a
therapeutically effective amount of the active substance with a
pharmaceutically acceptable carrier that can have different forms,
depending on the way of administration. Pharmaceutical compositions
can be prepared by using conventional pharmaceutical excipients and
methods of preparation. The forms for oral administration can be
capsules, powders or tablets where usual solid vehicles including
lactose, starch, glucose, methylcellulose, magnesium stearate,
di-calcium phosphate, mannitol may be added, as well as usual
liquid oral excipients including, but not limited to, ethanol,
glycerol, and water. All excipients may be mixed with
disintegrating agents, solvents, granulating agents, moisturizers
and binders. When a solid carrier is used for preparation of oral
compositions (e.g., starch, sugar, kaolin, binders disintegrating
agents) preparation can be in the form of powder, capsules
containing granules or coated particles, tablets, hard gelatin
capsules, or granules without limitation, and the amount of the
solid carrier can vary (between 1 mg to 1 g). Tablets and capsules
are the preferred oral composition forms.
[0168] Pharmaceutical compositions containing compounds of the
present invention may be in any form suitable for the intended
method of administration, including, for example, a solution, a
suspension, or an emulsion. Liquid carriers are typically used in
preparing solutions, suspensions, and emulsions. Liquid carriers
contemplated for use in the practice of the present invention
include, for example, water, saline, pharmaceutically acceptable
organic solvent(s), pharmaceutically acceptable oils or fats, and
the like, as well as mixtures of two or more thereof. The liquid
carrier may contain other suitable pharmaceutically acceptable
additives such as solubilizers, emulsifiers, nutrients, buffers,
preservatives, suspending agents, thickening agents, viscosity
regulators, stabilizers, and the like. Suitable organic solvents
include, for example, monohydric alcohols, such as ethanol, and
polyhydric alcohols, such as glycols. Suitable oils include, for
example, soybean oil, coconut oil, olive oil, safflower oil,
cottonseed oil, and the like. For parenteral administration, the
carrier can also be an oily ester such as ethyl oleate, isopropyl
inyristate, and the like. Compositions of the present invention may
also be in the form of microparticles, microcapsules, liposomal
encapsulates, and the like, as well as combinations of any two or
more thereof.
[0169] Examples of pharmaceutically acceptable disintegrants for
oral compositions useful in the present invention include, but are
not limited to, starch, pre-gelatinized starch, sodium starch
glycolate, sodium carboxymethylcellulose, croscarmellose sodium,
microcrystalline cellulose, alginates, resins, surfactants,
effervescent compositions, aqueous aluminum silicates and
crosslinked polyvinylpyrrolidone.
[0170] Examples of pharmaceutically acceptable binders for oral
compositions useful herein include, but are not limited to, acacia;
cellulose derivatives, such as methylcellulose,
carboxymethylcellulose, hydroxypropylmethylcellulose,
hydroxypropylcellulose or hydroxyethylcellulose; gelatin, glucose,
dextrose, xylitol, polymethacrylates, polyvinylpyrrolidone,
sorbitol, starch, pre-gelatinized starch, tragacanth, xanthane
resin, alginates, magnesium-aluminum silicate, polyethylene glycol
or bentonite.
[0171] Examples of pharmaceutically acceptable fillers for oral
compositions include, but are not limited to, lactose,
anhydrolactose, lactose monohydrate, sucrose, dextrose, mannitol,
sorbitol, starch, cellulose (particularly microcrystalline
cellulose), dihydro- or anhydro-calcium phosphate, calcium
carbonate and calcium sulfate.
[0172] Examples of pharmaceutically acceptable lubricants useful in
the compositions of the invention include, but are not limited to,
magnesium stearate, talc, polyethylene glycol, polymers of ethylene
oxide, sodium lauryl sulfate, magnesium lauryl sulfate, sodium
oleate, sodium stearyl fumarate, and colloidal silicon dioxide.
[0173] Examples of suitable pharmaceutically acceptable odorants
for the oral compositions include, but are not limited to,
synthetic aromas and natural aromatic oils such as extracts of
oils, flowers, fruits (e.g., banana, apple, sour cherry, peach) and
combinations thereof, and similar aromas. Their use depends on many
factors, the most important being the organoleptic acceptability
for the population that will be taking the pharmaceutical
compositions.
[0174] Examples of suitable pharmaceutically acceptable dyes for
the oral compositions include, but are not limited to, synthetic
and natural dyes such as titanium dioxide, beta-carotene and
extracts of grapefruit peel.
[0175] Suitable examples of pharmaceutically acceptable sweeteners
for the oral compositions include, but are not limited to,
aspartame, saccharin, saccharin sodium, sodium cyclamate, xylitol,
mannitol, sorbitol, lactose and sucrose.
[0176] Suitable examples of pharmaceutically acceptable buffers
include, but are not limited to, citric acid, sodium citrate,
sodium bicarbonate, dibasic sodium phosphate, magnesium oxide,
calcium carbonate and magnesium hydroxide:
[0177] Suitable examples of pharmaceutically acceptable surfactants
include, but are not limited to, sodium lauryl sulfate and
polysorbates.
[0178] Suitable examples of pharmaceutically acceptable
preservatives include, but are not limited to, various
antibacterial and antifungal agents such as solvents, for example
ethanol, propylene glycol, benzyl alcohol, chlorobutanol,
quaternary ammonium salts, and parabens (such as methyl paraben,
ethyl paraben, propyl paraben, etc.).
[0179] Suitable examples of pharmaceutically acceptable stabilizers
and antioxidants include, but are not limited to,
ethylenediaminetetriacetic acid (EDTA), thiourea, tocopherol and
butyl hydroxyanisole.
[0180] The compounds of the invention may also, for example, be
formulated as suppositories e.g., containing conventional
suppository bases for use in human or veterinary medicine or as
pessaries e.g., containing conventional pessary bases.
[0181] For percutaneous or mucosal external administration, the
compound of Formula I can be prepared in a form of an ointment or
cream, gel or lotion. Ointments, creams and gels can be formulated
using a water or oil base with addition of an appropriate
emulsifier or gelling agent Formulation of the present compounds is
especially significant for respiratory inhalation, wherein the
compound of Formula I is to be delivered in the form of an aerosol
under pressure. It is preferred to micronize the compound of
Formula I after it has been homogenised, e.g., in lactose, glucose,
higher fatty acids, sodium salt of dioctylsulfosuccinic acid or,
most preferably, in carboxymethyl cellulose, in order to achieve a
microparticle size of 5 .mu.m or less for the majority of
particles. For the inhalation formulation, the aerosol can be mixed
with a gas or a liquid propellant for dispensing the active
substance. An inhaler or atomizer or nebulizer may be used. Such
devices are known. See, e.g., Newman et al., Thorax, 1985,
40:61-676 Berenberg, M., J. Asthma USA, 1985, 22:87-92. A Bird
nebulizer can also be used. See also U.S. Pat. Nos. 6,402,733;
6,273,086; and 6,228,346.
[0182] For application topically to the skin, the agent of the
present invention can be formulated as a suitable ointment
containing the active compound suspended or dissolved in, for
example, a mixture with one or more of the following: mineral oil,
liquid petrolatum, white petrolatum, propylene glycol,
polyoxyethylene polyoxypropylene compound, emulsifying wax,
sorbitan monostearate, a polyethylene glycol, liquid paraffin,
polysorbate 60, cetyl esters wax, cetearyl alcohol,
2-octyldodecanol, benzyl alcohol, and water. Such compositions may
also contain other pharmaceutically acceptable excipients, such as
polymers, oils, liquid carriers, surfactants, buffers,
preservatives, stabilizers, antioxidants, moisturizers, emollients,
colorants, and odorants.
[0183] Examples of pharmaceutically acceptable polymers suitable
for such topical compositions include, but are not limited to,
acrylic polymers; cellulose derivatives, such as
carboxymethylcellulose sodium, methylcellulose or
hydroxypropylcellulose; natural polymers, such as alginates,
tragacanth, pectin, xanthan and cytosan.
[0184] As indicated, the compound of the present invention can be
administered intranasally or by inhalation and is conveniently
delivered in the form of a dry powder inhaler or an aerosol spray
presentation from a pressurized container, pump, spray or nebulizer
with the use of a suitable propellant, e.g.,
dichlorodifluoromethane, trichlorofluoromethane,
dichiorotetrafluoroethane, a hydrofluoroalkane such as
1,1,1,2-tetrafluoroethane (HFA 134AT'''') or
1,1,1,2,3,3,3-heptafluoropropane (HFA 227EA), carbon dioxide or
other suitable gas. In the case of a pressurized aerosol, the
dosage unit may be determined by providing a valve to deliver a
metered amount. The pressurized container, pump, spray or nebulizer
may contain a solution or suspension of the active compound, e.g.,
using a mixture of ethanol and the propellant as the solvent, which
may additionally contain a lubricant, e.g., sorbitan trioleate.
[0185] Capsules and cartridges (made, for example, from gelatin)
for use in an inhaler or insufflator may be formulated to contain a
powder mix of the compound and a suitable powder base such as
lactose or starch.
[0186] For topical administration by inhalation the compounds
according to the invention may be delivered for use in human or
veterinary medicine via a nebulizer.
[0187] The pharmaceutical compositions of the invention may contain
from 0.01 to 99% weight per volume of the active material.
[0188] A therapeutically effective amount of the compound of the
present invention can be determined by methods known in the art.
Since the compound of the present invention is more efficiently
delivered to the desired site than other compounds such as
azithromycin and clarithromycin, a lesser amount of the compound of
the present invention can be delivered (on a molar basis) compared
to the azithromycin or clarithromycin while still achieving the
same therapeutic effect. Thus, the table below serves only as a
guide. Broad and preferred effective amounts of the compound, a
pharmaceutically acceptable derivative thereof are shown in the
table below.
TABLE-US-00001 Amount of Compound of Formula (I), or
Pharmaceutically Acceptable Derivative Thereof (.mu.mol/kg body
weight/day) Broad Range from about 0.004 to about 4000 Narrower
from about 0.04 to about 400 Still Narrower from about 4 to about
400 Narrowest from about 12 to about 120
[0189] The efficacy of the present compounds can be assessed by any
method for assessing inflammation or anti-inflammatory effect.
There are many known methods for this purpose including without
limitation use of contrast ultrasound in conjunction with injection
of microbubbles, measurement of inflammatory cytokines (such as
TNF-.alpha., IL-1, IFN-.gamma.) measurement of activated immune
system cells (activated T cells, cytotoxic T cells specifically
recognizing the inflamed or transplanted tissue) as well as by
observation (reduction of oedema, reduction of erythema, reduction
of pruritus or burning sensation, reduction of body temperature,
improvement in function of the afflicted organ) as well as any of
the methods provided below.
[0190] Administration may be once a day, twice a day, or more
often, and may be decreased during a maintenance phase of the
disease or disorder, e.g. once every second or third day instead of
every day or twice a day. The dose and the administration frequency
will depend on the clinical signs, which confirm maintenance of the
remission phase, with the reduction or absence of at least one or
more preferably more than one clinical signs of the acute phase
known to the person skilled in the art.
[0191] Biological Assays
[0192] The therapeutic effect of compounds of the present invention
was determined in in vitro and in vivo experiments such as the
following.
[0193] The cytokines assayed in the biological examples, when
expressed at elevated amounts, are markers for inflammation and, in
the case of cell proliferation, granulocyte degranulation and lung
neutrophilia, the behaviors of these immune cells are also markers
for their activation and, therefore, inflammation. Consequently,
reduction of pro-inflammatory cytokine expression or secretion and
reduction in cell proliferation, degranulation or neutrophil
accumulation is a measure of a compound's anti-inflammatory
activity. Lung neutrophilia specifically serves as a model for
COPD.
[0194] A compound analyzed using the biological assays as defined
herein is considered to be "active" if it is better than a positive
control (i.e., azithromycin) in at least one inhibitory function
(i.e., inhibition of TNF-.alpha. or IL-6) after stimulation with al
least one stimulant (e.g., PMA or PHA). More preferably, an active
compound exhibits more than 50% inhibition in at least one
inhibitory function
[0195] Sample Preparation
[0196] Test substances used in in vitro experiments were dissolved
in dimethyl sulfoxide (DMSO) (Kemika, Croatia) at concentrations of
50 mM and 10 mM, and were further diluted to final concentrations
of 50 .mu.M and 10 .mu.M in 1 mL Dulbecco's modified Eagle medium
(DMEM) supplemented with 1% heat inactivated fetal bovine serum
(FBS), 1% L-glutamine, 50 U/mi penicillin, 50 .mu.g/ml streptomycin
and 2.5 .mu.g/mL Fungizone (amphotericin B). Media and all media
supplements were purchased from Gibco, Australia, except the FBS
was purchased from Sigma, USA.
[0197] Isolation of Peripheral Blood Leukocytes
[0198] Peripheral blood leukocytes (PBL) were obtained from venous
blood of healthy volunteers by sedimentation on 2% dextran T-500
(Amersham Biosciences, USA) and subsequent centrifugation of
leukocyte rich plasma.
[0199] Inhibition of proinflammatory cytokine production by
stimulated human peripheral blood leukocytes in vitro
[0200] Peripheral blood leukocytes (PBL), isolated as described
above, were seeded in a 48-well plate at a concentration of 3 to
5.times.10.sup.6 cell per well in culture medium consisting of RPMI
1640 medium (Institute of Immunology, Croatia) supplemented with
10% heat-inactivated fetal calf serum (FCS, Biowhittaker, USA), 100
U/ml penicillin (Gibco, Australia), 100 .mu.g/ml streptomycin
(Gibco, Australia) and 2 mM L-glutamine (Gibco, Australia), and
preincubated with the test compounds for 2 h at 37.degree. C., in
an atmosphere of 5% CO.sub.2, and 90% humidity. Then, stimuli
(Sigma, USA) were added to a final concentration of 2 .mu.g/mL
lipopolysaccharide (LPS), 1 .mu.g/mL phorbol 12-myristate
13-acetate (PMA) or 120 .mu.g/mL zymosan. Samples were incubated
overnight under conditions described above. At the end of
incubation, the supernatants were transferred to eppendorf tubes
and centrifuged for 10 min at 1500.times.g. Concentrations of human
TNF-.alpha., IL-1.beta., IL-6 and IL-8 were determined in cell
supernatants by sandwich ELISA, using capture and detection
antibodies (R&D Systems, Minneapolis, MN) according to the
manufacturer's recommendations.
[0201] Inhibition (as percentage) was calculated using the
following formula:
inhibition=(1-concentration of cytokines in sample/concentration of
cytokines in positive control).times.100.
[0202] The positive control refers to LPS-, PMA- or
zymosan-stimulated samples that were not preincubated with test
compounds.
TABLE-US-00002 TABLE 1 Percentage of inhibition of proinflammatory
cytokine production by stimulated PBL treated with compounds conc.
TNF-.alpha. IL-1.alpha. IL-6 IL-8 Example (uM) PMA LPS zymosan PMA
LPS zymosan PMA LPS zymosan PMA LPS zymosan azithromycin 10 0 36 6
0 28 20 0 15 20 0 0 0 50 0 20 64 0 48 75 0 40 75 0 0 0
clarithromycin 10 0 0 0 26 0 36 0 11 28 33 23 0 50 0 0 0 33 0 59 0
0 0 0 0 0 1 10 0 54 88 0 67 73 0 10 29 0 0 29 50 94 100 100 84 93
83 96 94 100 100 97 100 3 10 91 72 76 73 87 88 60 56 44 30 48 32 50
83 81 96 68 86 91 66 78 74 22 71 16 8 10 46 51 87 71 66 86 24 16 57
40 38 31 50 87 85 92 86 83 94 100 63 80 89 79 88 9 10 93 73 14 69
72 82 30 22 0 0 18 0 50 85 96 72 72 82 0 95 100 100 94 96 91 10 10
41 49 14 30 44 48 12 14 19 0 27 28 50 62 86 72 0 41 49 18 71 39 42
52 36 12 10 49 23 77 45 43 88 32 16 67 0 7 2 50 97 92 97 90 95 100
67 81 91 3 72 9 15 10 82 61 93 80 80 96 50 39 76 0 38 42 50 96 99
100 96 98 99 95 93 96 95 94 91 17 10 46 13 30 55 25 58 37 9 33 21 4
20 50 67 21 46 75 44 90 55 14 60 13 6 13 18 10 38 9 21 34 16 51 33
10 34 7 6 25 50 57 12 30 68 45 90 61 23 63 0 0 22 21 10 8 0 5 31 14
58 17 1 22 6 0 0 50 75 10 39 69 54 86 63 22 50 23 4 13 22 10 0 0 0
14 5 57 16 3 28 0 0 10 50 55 23 35 56 46 76 60 28 51 15 5 30 23 10
5 0 0 12 2 67 20 0 35 0 0 22 50 54 19 37 69 39 75 59 14 45 15 10 43
25 10 41 0 19 58 22 82 51 4 51 0 0 17 50 91 71 88 86 61 93 87 62 86
34 45 63 33 10 23 16 16 33 28 55 9 8 13 0 0 0 50 74 50 53 75 42 85
49 30 40 10 11 40 34 10 86 55 65 86 69 77 66 28 66 33 21 32 50 100
96 100 100 82 99 95 91 99 76 87 81 37 10 28 15 17 14 60 49 41 20 21
17 14 27 50 45 76 96 25 69 98 57 78 100 10 0 0 40 10 55 67 60 0 0 1
0 7 0 31 24 6 50 77 82 76 0 28 55 0 0 0 12 0 0 41 10 30 39 27 52 79
80 59 25 36 10 29 19 50 70 95 99 100 84 100 79 92 100 42 87 63 48
10 31 49 82 38 64 85 30 57 73 25 36 49 50 90 87 97 78 79 92 84 97
97 83 85 90 49 10 37 69 70 35 84 76 76 70 76 0 44 15 50 86 91 93 84
86 25 86 99 97 95 86 54 50 10 0 10 36 0 19 81 0 0 52 0 0 0 50 100
100 98 44 23 69 23 54 96 100 61 70 .sup. 51C 10 18 5 38 0 2 62 27 4
42 12 0 26 50 4 0 0 0 0 0 6 0 0 0 0 0 53 10 82 28 64 92 83 92 66 20
68 30 9 19 50 100 100 100 99 95 100 95 99 100 63 100 92 54 10 25 3
11 62 60 56 0 0 9 0 0 0 50 96 55 92 97 69 98 71 58 87 40 82 53 58
10 58 39 61 66 72 86 42 29 56 29 21 21 50 97 95 100 52 90 99 89 93
100 82 86 95 59 10 98 70 91 82 94 99 75 61 80 72 42 54 50 98 99 100
45 82 91 90 98 100 74 81 96 60 10 88 55 87 88 86 94 59 46 68 67 42
39 50 99 99 100 65 91 97 92 97 100 83 83 97 63 10 21 0 0 52 59 45
24 8 0 0 0 0 50 73 69 87 81 89 100 62 60 90 3 36 44 90 10 40 78 53
61 62 82 46 67 60 0 4 24 50 95 100 94 57 91 100 90 88 100 53 38 88
95 10 34 12 47 25 0 38 17 1 18 0 0 5 50 50 30 63 75 0 42 56 8 58 21
16 33 96 10 14 3 21 32 0 36 27 0 17 0 0 6 50 48 13 61 81 0 88 64 3
76 12 17 40 97 10 31 0 43 57 0 49 32 0 9 4 0 10 50 57 62 69 79 0 34
51 14 56 29 23 34
[0203] Isolation of Peripheral Blood Mononuclear Cells
[0204] Heparinized peripheral blood was obtained from healthy
donors, and peripheral blood mononuclear cells (PBMC) were isolated
by Histopaque 1077 (Sigma, USA) density centrifugation at
400.times.g for 30 minutes. Collected PBMC were centrifuged in
plasma at 400.times.g for 10 min, resuspended and washed in RPMI
1640 (Institute of Immunology, Croatia) by centrifugation.
[0205] Inhibition of production of T-cell specific cytokines, IL-2
and IL-5, by stimulated human peripheral blood mononuclear cells in
vitro
[0206] Peripheral blood mononuclear cells (PBMC), isolated as
described above, were seeded in a 48-well plate at a concentration
of 1.times.10.sup.6 cells per well in the RPMI culture medium
described above. Cells were stimulated with 10 .mu.g/mL
phytohaemagglutinin (PHA) (Sigma, USA) and incubated at 37.degree.
C., 5% CO.sub.2 in 90% humidity with tested compounds (10 and 50
.mu.M) for 3 days. Cytokine concentrations were determined in
supernatants by sandwich ELISA, using capture and detection
antibodies (R&D, USA) according to the manufacturer's
recommendations.
[0207] Inhibition (as percentage) was calculated using the
following formula:
% inhibition=(1-concentration of cytokines in sample/concentration
of cytokines in positive control).times.100.
[0208] The positive control refers to LPS-, PMA- or
zymosan-stimulated samples that were not treated with test
compounds.
TABLE-US-00003 TABLE 2 Percentage of inhibition of IL-2 and IL-5
production by stimulated PBMC treated with test compounds IL-2 IL-5
clarithromycin 10 .mu.M 0 13 50 .mu.M 3 47 Example 8 10 .mu.M 100
34 50 .mu.M 100 85 Example 49 10 .mu.M 51 59 50 .mu.M 100 81
[0209] Effects on Human T-Cell Proliferation in vitro
[0210] The influence of the various test compounds at two different
concentrations (50 .mu.M and 10 .mu.M) on cell proliferation of
human peripheral blood mononuclear cells (PBMC) was assessed.
[0211] Heparinized peripheral blood was obtained from healthy
donors, and PBMC were isolated by Histopaque 1077 (Sigma, USA)
density centrifugation at 400.times.g for 30 minutes.
5.times.10.sup.4 cells/well were cultured for 3 days in the RPMI
medium as described above in the presence (positive control) or
absence (negative control) of stimulators [PHA (2.5 .mu.g/mL)
(Sigma, USA), or both PMA (10 ng/mL) (Sigma, USA) and ionomycin
(500 ng/mL) (Calbiochem, USA)], and in the presence of the test
compounds, at 37.degree. C. in an atmosphere of 5% CO.sub.2 and 90%
humidity. The cells were pulsed with 1 .mu.Ci of .sup.3H-thymidine
(Amersham, USA) per well during the last 18 h of the culture, and
were harvested on the 96-well filter (Packard Bioscience, USA)
using a multiple cell harvester (Packard, USA). The incorporation
of .sup.3H-thymidine in activated cells were measured using
TopCount NXT (Packard, USA).
[0212] Inhibition (as percentage) was calculated using the
following formula:
% inhibition=(1-(.sup.3H)thymidine incorporation expressed in
counts per minute (cpm) in sample/(.sup.3H)thymidine incorporation
expressed in cpm in positive control).times.100
[0213] The positive control refers to LPS-, PMA- or
zymosan-stimulated samples that were not treated with test
compounds.
TABLE-US-00004 TABLE 3 Percentage of inhibition of proliferation of
stimulated PBMC cell line treated with test compounds PHA PMA +
iono. Example 50 .mu.M 10 .mu.M 50 .mu.M 10 .mu.M azithromycin 29 5
31 9 clarithromycin 7 0 14 9 1 99 95 96 90 2 41 35 31 1 3 75 2 81
17 8 100 96 99 37 9 100 91 100 17 10 98 71 100 3 12 99 33 96 29 14
99 23 91 24 15 99 68 98 53 17 70 0 49 3 21 80 0 74 0 31 99 23 91 24
34 99 66 87 0 48 100 93 100 81 49 99 44 100 24
[0214] Isolation of Granulocytes
[0215] Granulocytes were obtained from heparinized whole blood
using density gradiant centrifugation. Erythrocytes were sedimented
on 3% dextran T-500 (Amersham Pharmacia Biotech AB, Uppsala,
Sweden). Leukocytes were centrifuged on Ficoll (Amersham Pharmacia
Biotech AB, Uppsala, Sweden) for 35 min at 600 g at 20.degree. C.
The pellet of granulocytes was cleared from remaining erythrocytes
by a brief hypotonic lysis.
[0216] Inhibition of Degranulation of Granulocytes
[0217] 1.times.10.sup.6 granulocytes, isolated as described above,
were resuspended in RPMI-1640 medium (Institute of Immunology,
Croatia), and were incubated with 10 or 50 .mu.M of test compound
together with cytochalasine B (5 .mu.g/mL) for 2 h at 37.degree. C.
Degranulation then was induced by the addition of 0.1 .mu.M fMLP
(Sigma, USA) or 0.5 .mu.M A23187 (Calbiochem, USA). The activity of
free neutrophil elastase was determined with a chromogenic
substrate specific for human neutrophil elastase, such as
N-(methoxysuccinyl)-L-alanyl-L-alanyl-L-prolyl-L-valine
4-nitroanilide (Sigma Chemical Company, St Louis, Mo., USA).
Elastase activity, as a marker of primary granules, was assessed in
supernatant using a spectrophotometer at an absorbance of 405
nm.
[0218] Results are expressed as the percent of inhibition of
degranulation in unstimulated cells, and in stimulated cells
treated with fMLP or A23187.
TABLE-US-00005 TABLE 4 Percentage of inhibition of degranulation of
granulocytes stimulated with fMLP or A23187 fMLP A23187 0.1 .mu.M
0.5 .mu.M azithromycin 50 .mu.M 36 15 erythromycin 50 .mu.M 23 0
Example 8 10 .mu.M 52 10 50 .mu.M 72 24 Example 49 10 .mu.M 47 10
50 .mu.M 72 51
[0219] Accumulation in Granulocytes
[0220] 7.5.times.10.sup.6 granulocytes, isolated as described
above, were suspended in 3 mL of RPMI 1640 (Institute of
Immunology, Croatia) containing 10 .mu.M of tested macrolide.
Samples were incubated at 37.degree. C. for 180 min. Following
incubation, the samples were centrifuged through the layer of
Poly(dimethylsiloxane-co-diphenylsiloxane), dihydroxy
terminated-silicone oil (Aldrich Chemical Company, Milwaukee, USA).
The pellet was resuspended in 0.5% Triton X-100 (Sigma, St. Louis,
USA) in deionized water (MilliQ, Millipore Corporation, Bedford,
USA). The suspension was sonicated, proteins were precipitated with
acetonitrile, and the concentration of macrolide in the
supernatants was determined by liquid chromatography-mass
spectrometry (LC-MS).
[0221] Intracellular concentrations of the test compounds were
calculated from the mean cell number recovered after centrifuging
through silicone oil. According to the literature, about 1 million
neutrophils is considered to have a volume of 0.24 .mu.L (Vazifeh
et al., Antimicrob Agents Chemo. 1997; 41: 2099-2107). To estimate
the extent of macrolide accumulation, the intracellular to
extracellular concentration ratio (PE) was calculated, where E (due
to a large volume of incubation medium) was taken as constant (10
.mu.M). Results are expressed relative to azithromycin according to
the following equation: % of azithromycin uptake=(I/E of a
substance/I/E of azithromycin).times.100. PE values obtained for
azithromycin were 164.+-.10.
TABLE-US-00006 TABLE 5 Accumulation of test compounds in
granulocytes expressed relative to azithromycin uptake Uptake
Example (% azithromycin) azithromycin 100 3 15 8 42 9 412 48 125 49
>600 .sup. 51C 122
[0222] Cytotoxicity Assay Hep G2 and A549 Cell Lines
[0223] To determine whether the anti-inflammatory activity of the
test compounds was due to observed inhibition of cytokine
production and inhibition of proliferation in vitro, and was not a
consequence of cellular cytotoxicity, measurement of succinate
dehydrogenase activity in living cells was performed. Cells were
cultured for 24 h in RPM medium as described above at 37.degree. C.
the presence of the test compounds at concentrations of 50 .mu.M
and 12.5 .mu.M. MTT [3-(4,5-dimethylthiazole-2-yl)-2,5-diphenyl
tetrazolium bromide] (Promega, USA), a detection reagent, was then
added and the cultures were incubated for 0.5-2 h. The amount of
MTT-Formazan produced was determined using a spectrophotometer at
490 nm (Mosmann, J. Immunol. Methods, 1983, 65: 55-63).
[0224] The percentage of inhibition of cell growth was calculated
using the following formula:
% inhibition of cell growth=OD.sub.490 treated cells/OD.sub.490
nontreated cells.times.100.
TABLE-US-00007 TABLE 6 Percentage of inhibition of cell growth
after treatment with substances conc. Example (.mu.M) Hep G2 A549
azithromycin 12.5 0 0 50 0 0 clarithromycin 12.5 0 0 50 0 0 1 12.5
0 0 50 15 3 2 12.5 0 0 50 1 3 3 12.5 0 0 50 0 0 8 12.5 3 0 50 5 3 9
12.5 4 0 50 15 0 10 12.5 0 12 50 5 6 12 12.5 0 8 50 0 4 14 12.5 0 4
50 0 0 15 12.5 3 0 50 0 5 17 12.5 5 5 50 2 2 18 12.5 5 0 50 3 0 21
12.5 8 5 50 4 4 22 12.5 3 5 50 1 0 23 12.5 6 0 50 5 0 25 12.5 10 8
50 8 4 31 12.5 0 0 50 0 0 33 12.5 5 0 50 4 0 34 12.5 7 0 50 1 1 37
12.5 3 1 50 19 0 40 12.5 2 0 50 2 0 41 12.5 7 1 50 3 0 48 12.5 0 0
50 9 0 49 12.5 5 0 50 26 6 50 12.5 0 10 50 8 10 .sup. 51/C 12.5 15
0 50 7 0 53 12.5 0 9 50 2 12 54 12.5 1 0 50 2 0 73 12.5 0 0 50 0 0
77 12.5 5 3 50 3 2 78 12.5 0 5 50 0 11 79 12.5 0 0 50 0 0 80 12.5 1
0 50 1 0 81 12.5 0 1 50 0 0 82 12.5 1 4 50 0 0 83 12.5 0 16 50 0
18
[0225] Lipopolvsaccharide Induced TNF-.alpha. Production in Balb/cJ
Mice
[0226] Male Balb/cJ mice (Iffa Credo, France) weighing 25-33 g were
randomly grouped (n=7 in test group, 7 in positive control group, 4
in negative control group). Test compounds, as well as vehicle
[0.125% carboxymethyl-cellulose (Sigma, USA)], were administered to
the mice intraperitoneally. Test compounds were administered at a
dose of 10 mg/kg of body weight, in a volume of 10 mL/kg of body
weight. Thirty minutes later, a sterile solution of
lipopolysaccharide (LPS) (Sigma, USA) in saline was
intraperitoneally administered at a concentration and volume of 25
.mu.mL/mouse to each animal, except those in negative control.
Ninety minutes following administration, all animals were
sacrificed by puncturing a. carotis communis. The plasma
concentration of TNF-.alpha. was determined by sandwich ELISA,
using capture and detection antibodies (R&D, USA) according to
manufacturer's recommendations. Results are presented in the
following table as percent of inhibition of TNF-.alpha. production
in comparison to the positive control (stimulated, but untreated
animals).
TABLE-US-00008 TABLE 7 Percentage of inhibition of TNF-.alpha.
production in LPS stimulated Balb/cJ mice treated with test
compounds Example % inhibition azithromycin 54 clarithromycin 63 1
64 2 59 8 93 12 69 14 96 15 100 17 84 21 61 31 92 34 78 48 52 49 43
54 58 62 59 73 90 77 92 78 89 79 84 80 86 81 91 82 72 83 77
[0227] Phorbol 12-myristate 13-acetate Induced Ear Oedema in CD1
Mice
[0228] Male CD1 mice (Iffa Credo, France) weighing 30-40 g were
randomly grouped (n=6 in test group of which the untreated ear
served as negative control; 6 in positive control group which also
served as their own negative control group). Test compounds, as
well as vehicle (Trans-phase Delivery System, containing benzyl
alcohol 10%, acetone 40% and isopropanol 50%) (all from Kemika,
Croatia), were administered topically to the internal surface of
the left ear thirty minutes prior to administration of phorbol
12-myristate 13-acetate (PMA) (Sigma, USA). Test compounds were
administered at a dose of 100, 250 or 500 .mu.g/15 .mu.L/ear.
Thirty minutes later, 0.01% PMA solution in acetone was applied
topically to the inner surface of the left ear of each animal in a
volume of 12 .mu.L/ear. During the treatment and challenge
(stimulation), animals were anaesthetized with anaesthesia by
inhalation. Six hours following challenge, animals were euthanized
by intraperitoneal thiopental injection (PLIVA, Croatia). For
assessing the auricular oedema, 8 mm discs were cut out of left and
right auricular pinna and weighed. The degree of oedema was
calculated by subtracting the weight of 8 mm disc of the untreated
ear from that of the treated contralateral ear. The inhibition of
oedema in the treated animals, presented in the Table 8, is
expressed as percentage compared to control mice (0%).
TABLE-US-00009 TABLE 8 Percentage of inhibition of PMA induced ear
oedema in CD1 mice treated with test compounds Oedema Compound Dose
(% inhibition) azithromycin 100 .mu.g 0 250 .mu.g 83 500 .mu.g 90
Example 8 100 .mu.g 54 250 .mu.g 74 500 .mu.g 94
[0229] Lung Neutrophilia Induced by Bacterial Lipopolysaccharide in
Male BALB/cJ Mice
[0230] Male BALB/cJ mice (Iffa Credo, France), with average weight
.about.30 g were randomly grouped (n=7 in testing group, 8 in
positive control, 7 in negative control). Mice were administered
intraperitoneally (i.p.) a single dose of 5 or 2.5 mg of test
compound. Two hours after administration, 2 .mu.g of bacterial
lipopolysaccharide (LPS), dissolved in PBS in a volume of 60 .mu.L,
was intranasally administered to all experimental groups except the
negative control group, which received the same volume (60 .mu.L)
of vehicle PBS. Animals were sacrificed approximately 24 hours
after application of LPS in order to obtain bronchoalveolar lavage
fluid (BALF), which was used to determine concentrations of IL-6
and TNF-.alpha., absolute number of cells, and the percentage of
neutrophils in BALF. Results are expressed as percentage of
decrease of total cell number, relative number of neutrophils, and
TNF-.alpha. and IL-6 concentration in BALF of treated animals
compared to positive control (LPS stimulated, but untreated
animals).
TABLE-US-00010 TABLE 9 Percentage of decrease of total cell number,
relative number of neutrophils, TNF-.alpha. and IL-6 concentration
in BALF of treated animals % decrease Dose of total % decrease of
Example (mg) cell no. neutrophils TNF-.alpha. IL-6 azithromycin 5
77 37 66 72 clarithromycin 5 77 36 78 60 8 5 84 52 69 67 12 5 88 46
87 45 48 5 79 66 85 9 54 5 90 70 100 100 62 2.5 47 13 62 22
[0231] Results are expressed as percentage of decrease of total
cell number, relative number of neutrophils, and TNF-alpha and IL-6
concentration in BALF of treated animals compared to positive
control (LPS stimulated, but untreated animals).
[0232] Besides accumulation of inflammatory cells in BALF, the
extent and anatomic site of pulmonary inflammation induced by LPS
was assessed 24 hours after PBS or LPS exposure. Accumulation of
granulocytes and mononuclear cells in peribronchial (PB) and
perivascular (PV) lung tissue areas and in alveolar spaces was
monitored following sacrifice of the animals.
[0233] Challenge with LPS induced significant accumulation of both
granulocytes and mononuclear cells in lung tissue in comparison to
groups challenged with PBS (negative control). The tested compounds
significantly decreased accumulation of both granulocytes and
mononuclears in lung tissue (PB and PV).
[0234] Septic Shock Induced by Bacterial Lipopolysaccharide in Male
BALB/cJ Mice
[0235] Male BALB/cJ mice, (Iffa Credo, Lyon, France) 8 weeks old,
weighing .about.25 g were randomly grouped (n=10 in testing groups
and 15 in control group). Animals were primed with 4 .mu.g
bacterial lipopolysaccharide (LPS) that was dissolved in saline and
administered i.pl. After 18-24 hours animals were challenged by an
intravenous injection of 90 .mu.g LPS in a volume of 0.2 mL.
Animals were treated with the test compounds or the vehicle
(control) intraperitoneally. or per os 30 min prior to each
injection of LPS. Survival throughout 24 hours was monitored.
TABLE-US-00011 TABLE 10 Effect of compounds on survival of BALB/cJ
mice after LPS-induced septic shock. Compound Dose Survival (%)
azithromycin 1 mg/kg p.o. 30 10 mg/kg p.o. 60 100 mg/kg p.o. 70
Example 8 10 mg/kg i.p. 50 100 mg/kg p.o. 100
[0236] In all the tests, the compounds of the present invention
were found to be very active as anti-inflamrnatories and the
anti-inflammatory activity was found to be comparable to or greater
than that of the comparative macrolide compounds.
[0237] It is therefore clear that the compounds of formula (I),
which have anti-inflammatory activity, can be useful in both acute
and chronic treatment and in the prophylaxis of inflammatory
pathologies, especially of those pathologies associated with
altered cellular functionality of the neutrophils, for example
rheumatoid arthritis, vasculitis, glomerulonephritis, damage from
ischemic reperfusion, atherosclerosis, septic shock, ARDS, COPD and
asthma.
[0238] The therapeutically effective quantities will depend on the
age and on the general physiological condition of the patient, the
route of administration and the pharmaceutical formulation used.
The therapeutic doses will generally be between about 10 and 2000
mg/day and preferably between about 30 and 1500 ing/day.
[0239] The compounds of the present invention for use in treatment
and/or prophylaxis of the pathologies indicated above will
preferably be used in a pharmaceutical form suitable for oral,
rectal, sublingual, parenteral, topical, transdermal and
inhalational administration.
[0240] The present invention further relates to pharmaceutical
formulations containing a therapeutically effective quantity of a
compound of formula (I) or one of its salts mixed with a
pharmaceutically acceptable vehicle. The pharmaceutical
formulations of the present invention can be liquids that are
suitable for oral and/or parenteral administration, for example,
drops, syrups, solutions, injectable solutions that are ready for
use or are prepared by the dilution of a freeze-dried product but
are preferably solid or semisolid as tablets, capsules, granules,
powders, pellets, pessaries, suppositories, creams, salves, gels,
ointments; or solutions, suspensions, emulsions, or other forms
suitable for administration by the transdermal route or by
inhalation.
[0241] Depending on the type of formulation, in addition to a
therapeutically effective quantity of one or more compounds of
formula (I), they will contain solid or liquid excipients or
diluents for pharmaceutical use and possibly other additives
normally used in the preparation of pharmaceutical formulations,
such as thickeners, aggregating agents, lubricants, disintegrating
agents, flavorings and colorants.
[0242] The pharmaceutical formulations of the invention can be
produced in accordance with the usual methods.
[0243] In the following examples, the structural representation of
a pendant --O-- or --N is equivalent to the --OH, --NH, or
--NH.sub.2 as appropriate based on atom valency.
[0244] The following abbreviations are used in the text: DBU for
1,8-diazabicyclo[5.4.0]undec-7-ene, DCM for dichloromethane, DMAP
for 4-dimethylaminopyridine, DMF for N,N-dimethylformamide, DMSO
for dimethyl sulfoxide, EDC.times.HCl for
1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride, EtOAc
for ethyl acetate, KO-t-Bu for potassium tert-butoxide, MeOH for
methanol, EtOH for ethanol, t-BuOH for tert-butanol, TEA for
triethylamine and THF for tetrahydrofuran, MIBK for methyl
iso-buthyl ketone, DCC for dicyclohexylcarbodiimide, DMAP for
4-dimethylaminopyridine, DIPEA for N,N-diisopropylethylamine, CDI
for 1,1'-carbonyldiimidazole, and DCE for dichloroethane.
[0245] The compounds and process of the present invention will be
better understood in connection with the following examples, which
are intended as an illustration only and not limiting the scope of
the invention. Various changes and modifications to the disclosed
embodiments will be apparent to those skilled in the art and such
changes and modifications including, without limitation, those
relating to the chemical structures, substituents, derivatives
formulations and/or methods of the invention may be made without
departing from the spirit of the invention and the scope of the
appended claims.
EXAMPLES
[0246] 2'-O-Acetyl-protected compounds may be prepared by the
procedure described by W. R. Baker et al. in J. Org. Chem. 1988,
53, 2340 9-O-(2-chlorobenzyl)-protected oxime compounds may be
prepared by the procedure described by Y. Watanabe et al. in J.
Antibiot. 1993, 46, 1163. 9-O-(1-isopropoxycyclohexyl)-protected
oxime compounds may be prepared by the procedure described by Z. Ma
et al. in J. Med. Chem. 2001, 44, 4137. 11,12-Carbonate compounds
may be prepared by procedure as described in international patent
application WO 02/50091 or by procedure as described by S. Djokic
et al. in J. Chem. Res. (S) 1988, 152. 11-O-Methyl azithromycin may
be prepared by procedure as described by Kobrehel et al. in J.
Antibiotics 1992, 45, 527. All documents cited in this paragraph
are incorporated by reference in their entirety.
Example 1
11,12-Carbonate-11,12-dideoxy-4''-O-(3-diethylamino-propionyl)-azithromyci-
n
##STR00014##
[0248] 11,12-Carbonate-11,12-dideoxy-4''-O-propenoyl-azithromycin
(0.5 g, 0.6 mmol), obtained as described in international patent
application WO 03/042228, intermediate 50, and diethylamine (0.72
mL, 7 mmol) were dissolved in dry methanol (60 mL) and the mixture
stirred overnight at 40.degree. C. Methanol was evaporated under
reduced pressure and the crude product was purified by column
chromatography (DCM/MeOH/NH.sub.4OH=90:9:0.5) to afford the title
compound.
[0249] MS (ES+) m/z: [MH].sup.+=902.46
[0250] .sup.13C-NMR(125 MHz, CDCl.sub.3) .delta.: 177.1, 172.1,
153.3, 102.8, 95.3, 85.9, 85.1, 79.0, 78.0, 76.3, 73.3, 73.0, 70.7,
68.2, 67.7, 65.5, 62.9, 61.2, 49.5, 48.2, 46.7, 45.3, 43.0, 41.9,
40.4, 35.3, 34.4, 29.3, 26.8, 26.2, 22.1, 22.0, 21.6, 21.2, 17.8,
14.9, 14.2, 10.5, 10.4, 5.5.
Example 2
4''-O-(3-Diethylamino-propionyl)-8a-aza-8a-homoerythromycin A
##STR00015##
[0252] 4''-O-Propenyl-8a-aza-8a-homoerythromycin A (0.34 g, 0.42
mmol), obtained as described in international patent application WO
02/32917 (incorporated by reference) example 59, and diethylamine
(0.56 mL, 5.4 mmol) were dissolved in dry methanol (50 mL) and the
mixture stirred overnight at 40.degree. C. Methanol was evaporated
under reduced pressure and the crude product was purified by column
chromatography (DCM/MeOH/NH.sub.4OH=90:9:1.5) to afford the title
compound.
[0253] MS (ES+) m/z: [MH].sup.+=876.48
[0254] .sup.13C-NMR(125 MHz, DMSO) .delta.: 177.2, 174.6, 171.8,
102.2, 93.9, 82.1, 77.9, 76.5, 75.7, 74.6, 73.2, 72.5, 70.7, 70.4,
65.8, 64.7, 62.1, 48.7, 48.2, 45.9, 44.8, 42.2, 40.7, 40.2, 34.2,
32.7, 30.3, 27.2, 23.5, 21.5, 21.4, 20.5, 17.6, 17.2, 14.4, 11.5,
11.3, 9.2, 8.8.
Example 3
4''-O-Diethylamino-propionyl)-6-O-methyl-8a-aza-8a-homoerythromycin
A
##STR00016##
[0256] Diethyl amine (80 .mu.L, 0.95 mol) was added to a solution
of 4''-O-propenoyl-6-O-methyl-8a-aza-8a-homoerythromycin A (0.15 g,
0.19 mmol), obtained as described in international patent
application WO 02/32917, example 9, (0.15 g, 0.19 mmol) in
isopropanol (2 mL) and the reaction mixture was stirred at
70.degree. C. overnight in tube. Isopropanol was evaporated, the
residue was purified by SP column (DCM/MeOH/NH.sub.4OH=90:9:0.5) to
give the title compound (34 mg).
[0257] MS (ES) m/z: [MH].sup.+=890 (95%).
Example 4
2'-O-Acetyl-3'-N-methyl-3'-N-(2-cyanoethyl)-4''-O-propenoyl-6-O-methyl-9a--
aza-9a-homoerythromycin A
Intermediate 1: 3'-N-Demethyl-6-O-methyl-9a-aza-9a-homoerythromycin
A
[0258] In a solution of a 9a-lactame (WO 99/51616, incorporated by
reference) (1 g, 1.31 mmol) in methanol (25 ml) sodium acetate
(0.54 g, 6.56 mmol) and iodine (0.37 g, 1.44 mmol) were added. The
reaction mixture was kept stirring and irradiated with a 500 W for
8 hours, while reaction mixture refluxed. The methanol was
evaporated off under vacuum and the residue was taken up in ethyl
acetate and extracted with 10% sodium thiosulphate (3.times.20 ml).
The combined aqueous phase was treated with 0.1 N NaOH solution to
alkaline pH and extracted with ethyl acetate (4.times.20 ml). After
drying with sodium sulphate, the organic phase was filtered off and
evaporated under vacuum to give intermediate 1 (0.64 g, 66%
yield).
[0259] MS (ES) m/z: [MH].sup.+=748 (90.47%)
Intermediate 2:
3'-N-Methyl-3'-N-(2-cyanoethyl)-6-O-methyl-9a-aza-9a-homoerythromycin
A
[0260] A solution of intermediate 1 (0.64 g, 0.854 mmol in
acrylonitrile (20 ml) was refluxed over the night. The excess
acrylonitrile was evaporated off under vacuum to give the crude
product of the intermediate 2. Purification by SP column (silica,
eluent CH.sub.2Cl.sub.2:MeOH:NH.sub.3=90:9:0.5) gave intermediate 2
(0.43 g, 64% yield).
[0261] MS (ES) m/z: [MH].sup.+=802 (88%)
Intermediate 3:
2'-O-Acetyl-3'-N-methyl-3'-N-(2-cyanoethyl)-6-O-methyl-9a-aza-9a-homoeryt-
hromycin A
[0262] Acetic anhydride (41 .mu.l, 0.43 mmol) was added to a
solution of intermediate 2 (0.49 g, 0.6 mmol) and DIPEA (149 .mu.l,
0.86 mmol) in CH.sub.2Cl.sub.2 (5 ml) and the reaction mixture was
stirred overnight. The CH.sub.2Cl.sub.2 was evaporated under vacuum
and the residue was taken up in ethyl acetate and extracted with
NaHCO.sub.3. After drying with sodium sulphate, the organic phase
was filtered off and evaporated under vacuum to give intermediate 3
(0.36 g, 60% yield).
[0263] MS (ES) m/z: [MH].sup.+=844 (75%)
##STR00017## ##STR00018##
[0264] To a solution of intermediate 3 (0.362 g, 0.43 mmol) in dry
toluene (5 ml), TEA (238 .mu.l , 1.72 mmol) and 3-chloropropionyl
chloride (82 .mu.l, 0.86 mmol) were added. The reaction mixture was
cooled down with water (15-20.degree. C.) and stirred for 4 hours.
Toluene was evaporated under vacuum and the residue was extracted
with CH.sub.2Cl.sub.2 and NaHCO.sub.3. After drying with sodium
sulfate, the organic phase was filtered off and evaporated under
vacuum to give titled compound (0.36 g, 95% yield).
[0265] MS (ES) m/z: [MH].sup.+=898 (63%)
Example 5
2'-O-Acetyl-3'-N-methyl-3'-N-(2-cyanoethyl)-4''-O-(3-diethylamino-propiony-
l)-6-O-methyl-9a-aza-9a-homoerythromycin A
[0266] Diethyl amine (186 .mu.l, 1.78 mmol) was added to a solution
of compound of example 4 (0.32 g, 0.357 mmol) in isopropanol (5 ml)
and the reaction mixture was stirred at 70.degree. C. over night in
a screw cup. Isopropanol was evaporated giving crude titled
compound (0.09 g, 0.093 mmol).
Example 6
2'-O-Acetyl-3'-N-methyl-3'-N-(3-aminopropyl)-4''-O-(3-diethylamino-propion-
yl)-6-O-methyl-9a-aza-9a-homoerythromycin A
[0267] Compound from example 5 (0.09 g, 0.093 mmol) was dissolved
in glacial acetic acid (25 ml) PtO.sub.2 was added. Using Parr
apparatus, the solution was stirred under a hydrogen atmosphere
overnight. Filtration through a pad of Celite, evaporation under
vacuum and puriffication by SP column (5 g silica, eluent
CH.sub.2Cl.sub.2:MeOH:NH.sub.3=90:9:0.5) gave the title product
(16.5 mg).
[0268] MS (ES) m/z: [MH].sup.+=975 (88%)
Example 7
4'',11-di-O-(3-Diethylamino-propionyl)-6-O-methyl-9a-aza-9a-homoerythromyc-
in A
##STR00019##
[0270] Diethylamine (2.08 mL, 20 mmol) was added to the solution of
compound
2'-O-acetyl-4'',11-di-O-propenoyl-6-O-methyl-9a-aza-9a-homoeryth-
romycin A (prepared according to WO 03/042228 with excess of
3-chloropropionyl chloride) (0.91 g, 1 mmol) in dry methanol (100
mL). The reaction mixture was stirred overnight at 40.degree. C.
Methanol was evaporated under reduced pressure and the crude
product was purified by column chromatography
(DCM-MeOH-NH.sub.4OH=90:9:1.5) to afford the title compound (0.59
g, 58% yield) as a white solid.
[0271] MS (ES) m/z: [MH].sup.+=1018
Example 8
4''-O-(3-Diethylamino-propionyl)-6-O-methyl-9a-aza-9a-homoerythromycin
A
##STR00020##
[0273] Diethylamine (1.04 mL, 10 mmol) was added to the solution of
compound
2'-O-acetyl-4''-O-propenyl-6-O-methyl-9a-aza-9a-homoerythromycin A
(prepared according to WO 03/042228, incorporated by reference)
(0.86 g, 1 mmol) in dry methanol (100 mL) The reaction mixture was
stirred overnight at 40.degree. C. Methanol was evaporated under
reduced pressure and the crude product was purified by flash
chromatography (DCM/MeOH/NH.sub.4OH=90:9:0.5) to afford the title
compound (0.5 g, 57% yield) as a white solid.
[0274] MS (ES) m/z: [MH].sup.+=891
[0275] All compounds in this Example each have the substituent
bonded at the C-2 of the desosaminyl sugar in the S absolute
stereochemical configuration, and therefore, is in an anti
stereochemical configuration compared to the stereochemical
configurations of the substituents bonded at the C-1 and C-3 of the
desosaminyl sugar.
Example 9
11,12-Carbamate-11,12-dideoxy-4''-O-(3-diethylamino-propionyl)-6-O-methyl--
erythromycin A
##STR00021##
[0277] Diethylamine (1.04 mL, 10 mmol) was added to the solution of
11,12-carbarnate-11,12-dideoxy-4''-O-propenoyl-6-O-methyl-erythromycin
A (WO 03/042228) (0.83 g, 1 mmol) in dry methanol (100 mL). The
reaction mixture was stirred overnight at 40.degree. C. Methanol
was evaporated under reduced pressure and the crude product was
purified by column chromatography (silica,2000/200/8
CH.sub.2Cl.sub.2/hexane/TEA eluant) to afford the title compound
(0.58 g, 64% yield) as a white solid.
[0278] MS (ES) m/z: [MH].sup.+=901
Example 10
11,12-(N-Methyl-carbamate)-11,12-dideoxy-4''-O-(3-diethylamino-propionyl)--
6-O-methyl-erythromycin A
##STR00022##
[0280] Diethylamine (1.04 mL, 10 mmol) was added to the solution of
11,12-(N-methyl-carbamate)-11,12-dideoxy-4''-O-propenoyl-6-O-methyl-eryth-
romycin A (WO 03/042228) (0.88 g, 1 mmol) in dry methanol (100 mL).
The reaction mixture was stirred overnight at 40.degree. C.
Methanol was evaporated under reduced pressure and the crude
product was purified by column chromatography (silica, 90/9/0.5
CH.sub.2Cl.sub.2/MeOH/NH.sub.4OH eluent) to afford the title
compound (0.68 g, 75% yield) as a white solid.
[0281] MS (ES) m/z: [MH].sup.+=915
Example 11
4''-O-(3-methylamino-propionyl)-6-O-methyl-9a-aza-9a-homoerythromycin
A
##STR00023##
[0283] 4''-O-Propenyl-6-O-methyl-9a-aza-9a-homoerythromycin A
(prepared according to WO 03/042228) (0.82 g, 1 mmol) was dissolved
in methylamine (33% wt. solution in absolute ethyl alcohol (6 mL,
50 mmol). The reaction mixture was stirred for 4 hours at room
temperature. Ethanol was evaporated under reduced pressure and the
crude product was purified by column chromatography (silica,
90/9/1.5 CH.sub.2Cl.sub.2/MeOH/NH.sub.4OH eluent) to afford the
title compound (0.48 g, 57% yield) as a light yellow solid.
[0284] MS (ES) m/z: [MH].sup.+=849
Example 12
4''-O-(3-dimethylamino-propionyl)-6-O-methyl-9a-aza-9a-homoerythromycin
A
##STR00024##
[0286] Formaldehyde (37 wt % solution in water) (0.23 mL, 3 mmol)
and formic acid (0.055 mL, 1.5 mmol) was added to the solution of
compound from example 11 (0.85 g, 1 mmol) in chloroform (40 mL).
The reaction mixture was stirred for 3 hours at 70.degree. C.
Chloroform was evaporated under reduced pressure and the crude
product was purified by column chromatography (silica, 90/9/1.5
CH.sub.2Cl.sub.2/MeOH/NH.sub.4OH eluent) to afford the title
compound (0.62 g, 72% yield) as a white solid.
[0287] MS (ES) m/z: [MH].sup.+=863
Examples 13 to 44
[0288] General Procedure for Michael Addition
[0289] To a solution of 4''-O-Propenyl-9a-aza-9a-homoerythromycin
A, obtained as described in international patent application WO
02/32917 (incorporated by reference) in acetonitrile (1 mL) was
added 5 equivalents of the amine component. The mixture was heated
at 70.degree. C. for 48 hours, cooled to room temperature and a
scavanger resin (isocyanate polymer bound for secondary amities or
4-benzyloxybenzaldehyde polymer bound for primary amines, 3 eq.)
and CH.sub.2Cl.sub.2 (3 mL) were subsequently added. After 1 day
the resin was filtered off, washed with MeOH (1 mL),
CH.sub.2Cl.sub.2 (1 mL) and again with MeOH (1 mL). The solvent was
evaporated giving the desired product.
[0290] The table that follows the General procedure gives the
structures of amines, as well as the products of formula (I).
TABLE-US-00012 MS (ES+) m/z [MH].sup.+ (m/z Amine Product
[MH.sub.2].sup.2+) ##STR00025## ##STR00026## 932.6 (466.3)
##STR00027## ##STR00028## 1003.6 (501.8) ##STR00029## ##STR00030##
893.6 (446.8) ##STR00031## ##STR00032## ##STR00033## ##STR00034##
975.6 (487.8) ##STR00035## ##STR00036## 992.6 (496.3) ##STR00037##
##STR00038## 962.6 (481.3) ##STR00039## ##STR00040## ##STR00041##
##STR00042## 948.6 (474.3) ##STR00043## ##STR00044## 1029.6 (514.8)
##STR00045## ##STR00046## 877.6 (438.8) ##STR00047## ##STR00048##
992.8 (496.4) ##STR00049## ##STR00050## 931.6 (465.9) ##STR00051##
##STR00052## 1087.8 (543.9) ##STR00053## ##STR00054## ##STR00055##
##STR00056## 891.6 (445.8) ##STR00057## ##STR00058## ##STR00059##
##STR00060## 962.6 ##STR00061## ##STR00062## 1017.8 ##STR00063##
##STR00064## 1031.8 ##STR00065## ##STR00066## 1015.6 ##STR00067##
##STR00068## 1029.8 ##STR00069## ##STR00070## 946.6 ##STR00071##
##STR00072## 918.6 ##STR00073## ##STR00074## 931.8 ##STR00075##
##STR00076## 918.6 ##STR00077## ##STR00078## 903.6 ##STR00079##
##STR00080## 875.6 ##STR00081## ##STR00082## 917.8 ##STR00083##
##STR00084## 905.6 ##STR00085## ##STR00086## 903.8 ##STR00087##
##STR00088## 889.6
Example 45
3'-N-Methyl-3'-N-2'-O-diacetyl-4''-O-propenoyl-6-O-methyl-9a-aza-9a-homoer-
ythromycin A
##STR00089##
[0291] Intermediate 1
3'-N-Methyl-3'-N-2'-O-diacetyl-6-O-methyl-9a-aza-9a-homoerythromycin
A
[0292] Starting from intermediate 1 of Example 4 and acetic acid in
the presence of DCC and DMAP in dicloromethane intermediate 1 was
obtained.
[0293]
3'-N-Methyl-3'N-2'-O-diacetyl-4''-O-propenoyl-6-O-methyl-9a-aza-9a--
homoerythromycin A
[0294] In solution of intermediate 1 in dry toluene in the presence
of TEA and 3-chloropropionyl chloride, according to the procedure
described in WO 03/042228 the crude title compound was
obtained.
Example 46
3'-N-Methyl-3'-N-2'-O-diacetyl-4''-O-(3-diethylamino-propionyl)-6-O-methyl-
-9a-aza-9a-homoervthromycin A
##STR00090##
[0296] Starting from example 45 according to procedure of example
1, and after purification by column chromatography
(CH.sub.2Cl.sub.2:MeOH: NH.sub.3=90:9:0.5) the title compound was
obtained.
[0297] MS (ES+) m/z: [MH].sup.+=960.36
Example 47
3'-N-Methyl-3'-N-(3-aminopropyl)-4''-O-(3-Diethylamino-propionyl)-6-O-meth-
yl-9a-aza-9a-homoerythromycin A
[0298] Method A
[0299] The title compound was prepared according to a synthetic
route as shown in Scheme below in which amount of each intermediate
is specified together with molecular mass obtained by LC/MS
analysis.
##STR00091## ##STR00092##
[0300] Method B
##STR00093##
[0301] The compound of Example 61 (0.18 g, 0.19 mmol) was dissolved
in glacial HOAc (10 ml) and catalyst PtO.sub.2 (80 mg) was added.
The reaction mixture was hydrogenated at 5 bar and 25.degree. C.
for 12 h. The catalyst was separated by filtration and the filtrate
was poured into water (40 ml). The pH was adjusted to 8 with 10%
NaOH and the mixture was extracted with EtOAc (3.times.30 ml).
Combined organic layers where washed with saturated aqueous
solution of NaHCO.sub.3 (3.times.20 ml), brine (3.times.20 ml),
water (3.times.20 ml) and dried over MgSO.sub.4. After evaporation
of the solvent, the crude product was purified using Flashmaster
II--solid phase extraction techniques (SPE 5 g) to afford 30 mg of
the title product.
[0302] MS m/z: (ES): MH.sup.+=933.7
Example 48
4''-O-(3-diethylamino-propionyl)-azithromycin
##STR00094##
[0304] 4"-O-Propenyl-6-O-methylerithromycin (0.85 g, 1.05 mmol),
obtained as described in international patent application WO
03/042228, and diethylamine (0.6 mL, 6 mmol) were dissolved in
CH.sub.3CN (20 mL) and H.sub.2O (2 ml). The mixture was stirred
overnight at 65.degree. C. The solvents were evaporated under
reduced pressure and the crude product was purified by column
chromatography (DCM/MeOH/NH.sub.4OH=90:9:0.5) to afford the title
compound.
[0305] MS (ES+) m/z: [MH].sup.+=877.1
Example 49
4''-O-(3-Diethylamino-propionyl)-6-O-methylerithromycin A
##STR00095##
[0307] 4''-O-Propenyl-6-O-methylerythromycin (1.0 g, 1.2 mmol),
obtained as described in international patent application WO
03/042228 and diethylamine (0.6 mL, 6 mmol) were dissolved in
CH.sub.3CN (20 mL) and H.sub.2O (2 ml). The mixture was stirred
overnight at 60.degree. C. The solvents were evaporated under
reduced pressure and the crude product was purified by column
chromatography (DCM/MeOH/NH.sub.4OH=90:9:0.5) to afford the title
compound.
[0308] MS (ES+) m/z: [MH].sup.+=876.0
Example 50
4''-O-(3-Piperazin-1-yl-propionyl)-6-O-methylerithromycin A
##STR00096##
[0310] To a solution of 4''-O-propenyl-6-O-methylerithromycin (1.0
g), obtained as described in international patent application WO
03/042228, in acetonitrile (10 ml) were added piperazine (0.431 g,
5 mmol), water (1.14 ml) and triethylamine (0.455 ml) and
suspension was heated to 80.degree. C. for 2 hours. The solvent was
evaporated and the residue was extracted with EtOAc and water
(2.times.50 ml). Organic layer was washed with brine and
NaHCO.sub.3 (2.times.50 ml). The organic layer was dried over
K.sub.2CO.sub.3 and evaporated in vacuum yielding the title product
(1.0 g).
[0311] MS (ES+) m/z: [MH].sup.+=889.9
Examples 51A to C
##STR00097##
[0312] Example 51A
11,12-Carbonate-11,12-dideoxv-2'-O-acetyl-4''-O-(2-cyanoethyl)-azithromyci-
n
[0313] 2'-O-Acetyl-11,12-carbonate-11,12-dideoxy-azithromycin (10
g, 12 mmol), obtained as described in international patent
application WO 03/042228, was dissolved in acrylonitrile (100 ml)
in N.sub.2 atmosphere. t-BuOH (3.75 ml, 5.8 mmol) was added and the
reaction mixture was cooled in an ice bath (0.degree. C.). NaH (0.3
g, 12 mmol) was added in portions during 10 minutes and the
reaction mixture was stirred overnight at room temperature.
Acrylonitrile was evaporated under reduced pressure. To the residue
EtOAc (250 ml) was added, extracted with water (150 ml). Water
layer was washed with EtOAc (100 ml). Combined organic layers were
washed with water (200 ml), dried over K.sub.2CO.sub.3 and
evaporated under reduced pressure yielding 10.5 g of the
Intermediate 1 which was used in the next step without
purification.
[0314] MS; m/z (ES): 870.1 [MH].sup.+
Example 51B
11,12-Carbonate-11,12-dideoxy-2'-O-acetyl-4''-O(3-aminopropyl)-azithromyci-
n
[0315] A high pressure reactor was filled with solution of
Intermediate 1 from Example 51 A (5 g, 5.7 mmol) in acetic acid
(150 ml). PtO.sub.2 (1.6 g) was added and the reaction mixture was
stirred at 5 bar overnight. Catalyst was filtered through cellite
and solvent evaporated in vacuum yielding 12 g of crude the
product, which was used in the next step without purification.
[0316] MS; m/z (ES): 874 [MH].sup.+
Example 51C
11,12-Carbonate-11,12-dideoxy-4''-O-(3-aminopropyl)-azithromycin
[0317] Example 51B was dissolved in MeOH (250 ml) and stirred at
55.degree. C. for 24 hours (pH of the reaction mixture was adjusted
to 8 with NH.sub.3/H.sub.2O=1/1). The solvent was evaporated under
reduced pressure and to the residue DCM (50 ml) and water (50 ml)
were added and pH was adjusted to 6 with 0.25M HCl. Layers were
separated and organic layer was evaporated yielding 3 g of crude
product that was purified by column chromatography (fraction,
DCM:MeOH:NH.sub.3=90:15:1.5) yielding 2.5 g of the title
compound.
[0318] MS; m/z (ES): 832.1 [MH].sup.+
Example 52
11,12-Carbonate-11,12-dideoxv-4''-O-(3-dimethylamino-propyl)-azithromycin
[0319] To a solution of the compound of Example 51C (0.10 g, 0.12
mmol) in acetone (10 mL) formaldehyde (37 wt % solution in water)
(0.05 mL, 6 mmol) and formic acid (0.055 mL, 1.5 mmol) were added.
The reaction mixture was stirred for 4 hours at 40.degree. C.
Acetone was evaporated under reduced pressure and the crude product
was purified by column chromatography (silica, 90/9/1.5
CH.sub.2Cl.sub.2/MeOH/NH.sub.4OH eluent) to afford the title
compound as a white solid.
[0320] MS; m/z (ES): 860.54 [MH].sup.+.
Example 53
3''-N-Demethyl-4''-O-acryloyl-6-O-methyl-9a-aza-9a-homoerythromycin
##STR00098##
[0322] To a stirred solution of
4''-O-acryloyl-6-O-methyl-9a-aza-9a-homoerythromycin A (0.5 g, 0.61
mmol), obtained as described in international application WO
03/042228 intermediate 43, and sodium acetate trihydrate (0.23 g,
2.8 mmol) in methanol (12.5 ml), solid iodine (0.155 g, 0.61 mmol)
was added. The reaction mixture was irradiated with a 500 W halogen
lamp for 2 h, cooled to room temperature and the solvent was
evaporated. Solid residue was dissolved in ethyl acetate (100 ml),
filtered, and the filtrate was washed with saturated NaHCO.sub.3
(25 ml) and saturated NaCl (25 ml). The organic layer was dried
over Na2SO.sub.4 and evaporated to give 0.4 g of the title
compound.
[0323] MS (ES+) m/z: [MH].sup.+=803.4
[0324] .sup.13C NMR (125 MHz, CDCl.sub.3) .delta.: 179.6, 177.5,
131.6, 128.1, 101.5, 95.5, 79.4, 78.8, 78.5, 78.4, 76.0, 74.9,
74.3, 72.9, 72.8, 67.5, 63.3, 59.9, 51.5, 49.5, 45.4, 44.5, 41.2,
40.1, 37.7, 35.8, 35.1, 33.2, 21.6, 21.1, 20.8, 20.6, 19.6, 18.2,
16.2, 15.2, 14.0, 11.2, 9.8.
Example 54
3''-N-Demethyl-4''-O-(3-diethylaminopropionyl)-6-O-methyl-9a-aza-9a-homoer-
ythromycin A
##STR00099##
[0326] A solution of the compound of Example 53 (0.2 g, 0.25 mmol)
in diethyl amine (5 ml) was heated at 40.degree. C. overnight,
cooled to r.t, and evaporated to dryness. The residue was purified
by flash cromatography (hexane/ethylacetate/diethylamine=1:1:0.2)
to afford 0.21 g of the title compound.
[0327] MS (ES+) m/z: [MH].sup.+=876.5
[0328] .sup.13C NMR (125 MHz, CDCl.sub.3) .delta.: 179.2, 177.1,
101.0, 95.1, 78.9, 78.1, 78.0, 77.9, 75.6, 74.5, 73.9, 72.5, 72.4,
67.0, 62.7, 59.5, 51.1, 49.0, 48.1, 46.3, 45.0, 44.1, 40.8, 39.7,
36.9, 35.4, 34.6, 32.7, 32.3, 21.2, 20.7, 20.3, 20.2, 19.2, 17.8,
15.8, 14.7, 13.5, 11.3, 10.7, 9.3.
Example 55
4''-O-(4-Diethylaminobutanoyl)-6-O-methyl-9a-aza-9a-homoerythromycin
A
##STR00100##
[0330] A solution of
2'-O-acetyl-6-O-methyl-9a-aza-9a-homoerithromycin (0.8 g, 1 mmol)
obtained according to procedure described in internationale patent
application WO 03/042228, intermediate 41, diethylaminobutyric acid
(0.83 g, 5.22 mmol), EDC.times.HCl (2 g, 10 mmol), DMAP (1.2 g, 10
mmol) in dichloromethane (10 ml) was stirred overnight at r.t.
Solvent was evaporated, the residue dissolved in methanol (25 ml),
and stirred at r.t. for 48 h. Methanol was evaporated and the crude
product purified using Flashmaster II--solid phase extraction
technique (SPE 10 g, DCM/MeOH/NH.sub.4OH=90:5:0.5 as eluent) to
afford 0.24 g of the title product.
[0331] MS m/z: (ES): MH.sup.+=904.9
Example 56
4''-O-(2-Diethylaminoethanoyl)-6-O-methyl-9a-aza-9a-homoerythromycin
A
##STR00101##
[0333] A solution of
2'-O-acetyl-6-O-methyl-9a-aza-9a-homoerithromycin (0.5 g, 0.62
mmol), diethylaminoacetic acid (0.49 g, 3.72 mmol), EDC.times.HCl
(0.74 g, 3.72 mmol), DMAP (0.45 g, 3.72 mmol) in dichloromethane
(10 ml) was stirred overnight at r.t. Solvent was evaporated, the
residue dissolved in methanol (25 ml), and stirred at r.t. for 48
h. Methanol was evaporated and the crude product purified using
Flashmaster II--solid phase extraction technique (SPE 10 g,
DCM/MeOH/NH.sub.4OH=90:5:0.5 as eluent) to afford 0.27 g of the
title product.
[0334] MS m/z: (ES): MH.sup.+=876.4
Example 57
3'-N-methyl-3'-N-i-propyl-4''-O-(3-diethylaminopropionyl)-6-O-methyl-9a-az-
a-9a-homoerythromycin A
##STR00102##
TABLE-US-00013 [0335] purity yield Product R MS (ES, m/z) % %
Example 57 ##STR00103## 918.7 [M + H].sup.+, calculated 918.2 98.8
40.1 Example 58 ##STR00104## 904.7 [M + H].sup.+, calculated 904.2
95.1 42.9 Example 59 ##STR00105## 932.7 [M + H].sup.+, calculated
932.2 99.3 40.9 Example 60 ##STR00106## 918.8 [M + H].sup.+,
calculated 918.2 98.6 32.9
[0336] To the solution of the compound from Example 54 (0.05 g,
0.057 mmol) in CH.sub.3CN (4.0 ml), DIPEA (101 .mu.l, 0.570) and
i-propyliodide (0.2 g, 1.14 mmol) were added. The reaction mixture
was stirred for 24 h at 65.degree. C. After evaporation of the
solvent, the crude product was purified using Flashmaster II--solid
phase extraction technique (SPE 5 g) to afford 21.0 mg (40.1%) of
the title product.
Examples 58 to 60
[0337] General Procedure for 3'-N-alkylation
[0338] To the solution of compound from Example 54 (0.10 g, 0.114
mmol) in CH.sub.3CN (4.0 ml), DIPEA (25.4 .mu.l, 0.142), and
alkyl-iodide (0.28 mmol) were added. The reaction mixture was
stirred for 24 h at room temperature. After evaporation of the
solvent, the crude product was purified using Flashmaster II--solid
phase extraction technique (SPE 5 g).
Example 61
340
-N-methyl-3'-N-(2-cyanoethyl)-4''-(3-diethylaminopropionyl)-6-O-methyl-
-9a-aza-9a-homoeritromicin A
##STR00107##
[0340] Compound from Example 54 (0.20 g, 0.068 mmol) was dissolved
in acrylonitrile (5 ml). The reaction mixture was stirred for 24 h
at 60.degree. C. After evaporation of the solvent, the crude
product was purified using Flashmaster II--solid phase extraction
techniques (SPE 5 g). to afford 180 mg of the title product.
[0341] MS m/z: (ES): MH.sup.30 =929.2
Example 62
3'-N-methyl-3'-N-acetyl-4''-O-(3-diethylaminopropionyl)-6-O-methyl-9a-aza--
9a-homoerythromycin A
##STR00108##
[0343] A solution of compound from Example 54 (0.43 g, 0.49 mmol),
acetic anhydride (48 .mu.l, 0.51 mmol) and triethylamine (70 .mu.l,
0.25 mmol) in DCM (8 ml) was stirred for 2 h at room temperature.
The reaction mixture was diluted with DCM (150 ml), washed with
saturated aqueous NaHCO.sub.3 solution and brine, than dried over
Na.sub.2SO.sub.4. The product was purified using Flash-Si SPE
(flashmaster, 70 ml) and ethyl acetate/hexane/diethylamine=5:5:1 as
eluent to afford 0.32 g of the title product.
[0344] MS (ES+) m/z: [MH].sup.+=918.7
Example 63
3'-N-methyl-3'-N-propionyl-4''-O-(3-diethylaminopropionyl)-6-O-methyl-9a-a-
za-9a-homoerythromycin A
##STR00109##
[0346] A solution of compound from Example 54 (0.1 g, 0.11 mmol),
propionic anhydride (15 .mu.l, 0.11 mmol), and triethylamine (16
.mu.l, 0.11 mmol) in DCM (3 ml) was stirred for 2 h at room
temperature. The reaction mixture was diluted with DCM (150 ml),
washed with saturated aqueous NaHCO.sub.3 solution and brine, than
dried over Na.sub.2SO.sub.4. The product was purified using
Flash-Si SPE (flashmaster, 70 ml) and ethyl
acetate/hexane/diethylamine=5:5:1 as eluent to afford 0.07 g of the
title product.
[0347] MS (ES+) m/z: [MH].sup.+=932.7
Example 64
11,12-carbonate-11,12-dideoxy-2'-O-acetyl-4''-O-(3-diethylaminopropyl)-6-O-
-methylerythromycin A 9-oxime
[0348] Intermediate 1:
11,12-carbonate-11,12-dideoxy-2'-O-acetyl-4''-O-(2-cyanoethoxy)-6-O-methy-
lerythromycin A 9-[O-(2-chlorobenzyl)]oxime
##STR00110##
[0349] A solution of 11,12-carbonate-11,12-dideoxy-2'
-O-acetyl-6-O-methylerythromycin A 9-[O-(2-chlorobenzyl)]oxime (1.0
g, 1.05 mmol), t-BuOH (0.303 ml, 3.15 mmol) and NaH (46.2 mg, 1.15
mmol) in acrylonitrile (22 ml) was stirred for 6 h at 0.degree. C.
and then allowed to warm up to room temperature. Acrylonitrile was
evaporated, the residue dissolved in ethyl acetate, and filtrated
off. The filtrate was washed with water (3.times.20 ml) and brine
(3.times.20 ml), and dried over K.sub.2CO.sub.3. Evaporation of the
solvent afforded 1.49 g of the title product.
[0350] MS (ES+) m/z: [MH].sup.+=1008.4
Intermediate 2:
11,12-carbonate-11,12-dideoxy-2'-O-acetyl-4''-O-(3-aminopropyl)-6-O-methy-
lerythromycin A 9-[O-(2-chlorobenzyl)]oxime
##STR00111##
[0352] A suspension of Intermediate 1 (1.49 g, 1.48 mmol) and
PtO.sub.2 (250 mg) in glacial HOAc (50 ml) was hydrogenated at 5
bar overnight. The reaction mixture was filtrated, acetic acid
evaporated, the residue dissolved in DCM (30 ml) and H.sub.2O (30
ml) and pH adjusted to 9.4. Layers were separated, water layer
extracted with DCM (2.times.30 ml) and combined organic layers were
dried over K.sub.2CO.sub.3. Evaporation of DCM afforded 0.98 g of
the title product.
[0353] MS (ES+) m/z: [MH].sup.+=1012.5
Intermediate 3:
11,12-carbonate-11,12-dideoxy-2'-O-acetyl-4''-O-(3-diethylanoproyl)-6-O-m-
ethylerythromycin A 9-[O-(2-chlorobenzyl)]oxime
##STR00112##
[0355] A solution of Intermediate 2 (0.4 g, 0.4 mmol), acetaldehyde
(66.7 .rho.l, 1.19 mmol), NaBH(OAc).sub.3 (0.25 g, 1.19 mmol) and
ZnCl.sub.2 (54 mg, 0.4 mmol) in dichlorethane (20 ml) was stirred
at r.t. for 4 h. Reaction mixture was filtrated, dichloroethane
evaporated, the residue dissolved in DCM (20 ml) and H.sub.2O (20
ml), pH adjusted to 9.3, layers were separated, and water layer was
extracted with DCM (2.times.15 ml) and combined organic layers were
dried over K.sub.2CO.sub.3. Evaporation of DCM afforded 0.37 g of
the title product.
[0356] MS (ES+) m/z [MH].sup.+=1068.4
11,12-carbonate-11,12-dideoxy-2'-O-acetyl-4''-O-(3-diethylaminopropyl)-6-O-
-methylerythromycin A 9-oxime
##STR00113##
[0358] A suspension of Intermediate 3 (0.37 g, 0.35 mmol), glacial
HOAc (100 .mu.l, 1.75 mmol) and 10% Pd/C (150 mg) in MeOH (40 ml)
was hydrogenated at 5 bar overnight. The reaction mixture was
filtrated, pH adjusted to 5.5, fresh 10% Pd/C (185 mg) was added
and the reaction mixture hydrogenated at 70 bar for 3 days. The
catalyst was filtered off, solvent evaporated, the residue
dissolved in DCM (30 ml) and H.sub.2O (30 ml), pH adjusted to 9.4,
layers separated, water layer extracted with DCM (2.times.30 ml)
and combined organic layers dried over K.sub.2CO.sub.3. Evaporation
of DCM afforded 0.18 g of the title product.
[0359] MS (ES+) m/z: [MH].sup.+=944.9
Example 65
4''-O-(3-diethylaminopropyl)-6-O-methylerythromycin A 9-oxime
##STR00114##
[0361] A solution of the compounds of Example 64 (0.18 g, 0.19
mmol) and K.sub.2CO.sub.3 (0.45 g, 3.25 mmol) in MeOH (15 ml) and
H.sub.2O (5 ml) was stirred overnight at 50.degree. C. Methanol was
evaporated, ethyl acetate (20 ml) and H.sub.2O (10 ml) were added.
Layers were separated and organic layer dried over K.sub.2CO.sub.3.
Evaporation of the solvent afforded 0.167 g of the crude title
product which was purified using column chromatography on silica
gel (DCM/MeOH/NH.sub.4OH=90:9:0.5 as eluent) to obtain 90 mg of the
title product.
[0362] MS (ES+) m/z: [MH].sup.+=877.0
Example 66
4''-O-(3-diethylaminopropyl)-6-O-methylerythromycin A
##STR00115##
[0364] To a solution of the compound of Example 65 (50 mg, 0.06
mmol) and HCOOH (6.3 .mu.l, 0.168 mmol) in EtOH (0.7 ml) and
H.sub.2O (0.7 ml), Na.sub.2S.sub.2O.sub.5 (46 mg, 0.12 mmol) was
added at r.t. The reaction mixture was warmed up to 80.degree. C.
and second portion of Na.sub.2S.sub.2O.sub.5 (46 mg, 0.12 mmol) was
added and stirred for further 4 h at 80.degree. C. Ethanol was
evaporated, the residue diluted with DCM (25 ml) and water (25 ml),
pH adjusted to 9.3, layers separed, and water layer extracted with
DCM (2.times.10 ml). Combined organic layers were dried over
K.sub.2CO.sub.3. Evaporation of DCM afforded 0.18 g of the title
product.
[0365] MS (ES+) m/z: [MH].sup.+=861.5
Example 67
11,12-carbonate-11,12-dideoxy-2'-O-acetyl-4''-O-(3-aminopropyl)-6-O-methyl-
erythromycin A 9-[O-(1-isopropoxycyclohexyl)]oxime
Intermediate
11,12-carbonate-11,12-dideoxy-2'-O-acetyl-4''-O-(2-cyanoethyl)-6-O-methyl-
erythromycin A 9-[O-(1-isopropoxycyclohexyl)]oxime
##STR00116##
[0367] A solution of
11,12-carbonate-11,12-dideoxy-2'-O-acetyl-6-O-methylerythromycin A
9-[O-(1-isopropoxycyclohexyl)]oxime (1.7 g, 1.75 mmol), t-BuOH
(0.505 ml, 5.25 mmol) and NaH (77 mg, 1.925 mmol) in acrylonitrile
(36.6 ml) was stirred for 6 h at 0.degree. C., allowed to warm up
to room temperature and further stirred overnight. After
eavaporation of acrylonitrile the residue was dissolved in ethyl
acetate and filtrated off. Obtained filtrate was washed with water
(3.times.20 ml) and brine (3.times.20 ml) and dried over
K.sub.2CO.sub.3. Evaporation of the solvent afforded 2.07 g of the
title product.
[0368] MS (ES+) m/z: [MH].sup.+=1024.9
11,12-carbonate-11,12-dideoxy-2'-O-acetyl-4''-O-(3-aminopropyl)-6-O-methyl-
erythromycin A 9-oxime
##STR00117##
[0370] A suspension of the Example 67, Intermediate (2.07 g, 2.02
mmol) and PtO.sub.2 (360 mg) in glacial HOAc (65 ml) was
hydrogenated at 5 bar overnight. The reaction mixture was
filtrated, HOAc evaporated and the residue dissolved in DCM (30 ml)
and H.sub.2O (30 ml), pH was adjusted to 9.4, layers were
separated, and the water layer was extracted with DCM (2.times.30
ml). Combined organic layers were dried over K.sub.2CO.sub.3.
Evaporation of solvent afforded 1.35 g of the title product.
[0371] MS (ES+) m/z: [MH].sup.+=888.9
Example 68:
4''-O-(3-aminopropyl)-6-O-methylerythromycin A 9-oxime
##STR00118##
[0373] A solution of the compound of Example 67 (1.34 g, 1.51 mmol)
and K.sub.2CO.sub.3 (3.54 g, 25.7 mmol) in MeOH (100 ml) and
H.sub.2O (30 ml) was stirred at 50.degree. C. for 2 h and then
overnight at room temperature. After which MeOH was evaporated, DCM
(100 ml) and H.sub.2O (30 ml) were added. Layers were separated and
organic layer dried over K.sub.2CO.sub.3. Evaporation of the
solvent afforded 0.96 g of the crude title product which was
purified using column chromatography on silica gel
(DCM/MeOH/NH.sub.4OH=90:9:1.5 as eluent) to obtain 0.46 g of the
title product.
[0374] MS (ES+) m/z: [MH].sup.+=820.8
Example 69
4''-O-(3-aminopropyl)-6-O-methylerythromycin A
##STR00119##
[0376] To a solution of the compound of Example 68 (0.39 g, 0.48
mmol) and HCOOH (50.7 .mu.l, 1.34 mmol) in EtOH (5 ml) and H.sub.2O
(6 ml), Na.sub.2S.sub.2O.sub.5 (0.365 g, 0.96 mmol) was added at
room temperature. The reaction mixture was warmed up to 80.degree.
C. and than second portion of Na.sub.2S.sub.2O.sub.5 (0.365 g, 0.96
mmol) was added. After stirring at 80.degree. C. for 4 h, ethanol
was evaporated, the residue diluted with DCM (50 ml) and water (25
ml), pH adjusted to 9.3, layers separated, and water layer
extracted with DCM (2.times.10 ml). Combined organic layers were
dried over K.sub.2CO.sub.3. Evaporation of the solvent afforded
0.36 g of the crude product which was purified using column
chromatography on silica gel (DCM/MeOH/NH.sub.4OH=90:9:1.5 as
eluent) to obtain 0.22 g of the title product.
[0377] MS (ES+) m/z: [MH].sup.+=805.4
Example 70
11,12-carbonate-2'-O-acetyl-4''-O-(2-cyanoethyl)-6-O-methyl-9a-homoervthro-
mycin A
##STR00120##
[0379] A solution of
11,12-carbonate-2'-O-acetyl-6-O-methyl-9a-aza-9a-homoerythromycin A
(prepared according to WO 02/50091, incorporated by reference)
(1.68 g, 2.0 mmol) and tert-butanol (1 ml) in acrylonitrile (40 ml)
was cooled to 0.degree. C. Sodium hydride (0.15 g, 3.75 mmol, 60%
in oil) was then added and the reaction stirred for 7 h.
Acrylonitrile was evaporated under reduced pressure, ethyl acetate
(200 ml) was added and filtrated. Filtrate was washed with
saturated aqueous solution of NaHCO.sub.3 (100 ml) and brine (100
ml), and dried over anhydrous Na.sub.2SO.sub.4. Evaporation of the
solvent afforded crude product which was purified using column
chromatography on silica gel (DCM/MeOH/NH.sub.4OH=90:5:0.5 as
eluent) to obtain 1.33 g of the title product.
[0380] MS m/z: (ES): MH.sup.+=884.9
Example 71
4''-O-(2-cyanoethyl)-6-O-methyl-9a-aza-9a-homoerythromycin A
##STR00121##
[0382] A solution of the compound of Example 70 (1.33 g, 1.5 mmol)
and K.sub.2CO.sub.3 (0.45 g, 3.2 mmol) in MeOH (65 ml) and of
H.sub.2O (22 ml) was stirred for 7 h at 50.degree. C. Methanol was
then evaporated, EtOAc (200 ml) was added and layers separated.
Organic layer was washed with saturated aqueous solution of
NaHCO.sub.3 (100 ml) and brine (100 ml), and dried over anhydrous
Na.sub.2SO.sub.4. Evaporation of the solvent afforded 1.11 g of the
title product.
[0383] MS m/z: (ES): MH.sup.+=816.9
Example 72
4''-O-(3-aminopropyl)-6-O-methyl-9a-aza-9a-homoerytromycin A
##STR00122##
[0385] A solution of the compound of Example 71 (1.11 g, 1.32 mmol)
and PtO.sub.2 (0.25 g, 0.11 mmol) in glacial HOAc (60 ml) was
hydrogenated at 4.5 bar overnight. Catalyst was removed by
filtration and filtrate was washed with DCM (2.times.20 ml).
Filtrate was concentrated in vacuo affording the crude product
which was dissolved in DCM (100 ml), to which water was added (50
ml), pH was adjusted to 9.5, and layers separated. Organic layer
was dried over anhydrous Na.sub.2SO.sub.4. Evaporation of the
solvent afforded crude product which was purified using column
chromatography on silica gel (EtOAc/hexane/diethylamine 5:5:1 as
eluent) to obtain 0.6 g of the title product.
[0386] MS m/z: (ES): MH.sup.+=820.8
Example 73
4''-O-(3-Diethylaminopropyl)-6-O-methyl-9a-aza-9a-homoerythromycin
A
##STR00123##
[0388] A solution of the compound of Example 72 (100.0 mg, 0.12
mmol) in DCE (5 nil) was stirred for 5 min. at the room
temperature. Molecular sieves (4A), acetaldehyde (20.5 .mu.l, 0.36
mmol), NaBH(OAc).sub.3, (77.3 mg, 0.36 mmol) and ZnCl.sub.2 (16.6
mg, 0.12 mmol) were then added. The reaction mixture was stirred
for 1 hour at room temperature. After evaporation of the solvent,
the residue was taken up into DCM (10 ml) and extracted with
saturated aqueous solution of NaHCO.sub.3 (3.times.5 ml), brine
(3.times.5 ml) and water (3.times.5 ml). Combined aqueous layers
were extracted with DCM (2.times.20 ml). Combined DCM layers were
dried over MgSO.sub.4. After evaporation of the solvent, crude
product was purified using preparative HPLC (XTerra Prep RP 18
kolona 5 .mu.m 19.times.100 mm) to afford 26 mg of the title
product.
[0389] MS m/z: (ES): MH.sup.+=876.6
[0390] .sup.13C NMR (125 MHz. CDCl.sub.3) [.delta./ppm]
[0391] 179.9, 177.4, 102.3, 95.5, 87.8, 79.6, 78.5, 78.3, 76.2,
74.2, 73.6, 73.0, 72.9, 71.2, 67.7, 64.9, 64.9, 51.4, 49.7, 49.5,
46.8, 45.4, 44.7, 41.3, 40.1, 40.3, 35.6, 35.5, 29.2, 27.7, 21.8,
21.6, 20.7, 20.5, 19.4, 18.8, 16.2, 15.2, 14.0, 11.2, 9.2,
11.4.
Example 74
2'-O-Acetyl-4''-O-(2-cyanoethyl)-11-O-methyl-azithromyein
##STR00124##
[0393] To a solution of the compound of
2'-O-Acetyl-11-O-methyl-azithromycin (1.0 g, 1.2 mmol) in
acrylonitrile (25 ml), t-BuOH (0.3 ml, 3.6 mmol) and NaH (52.8 mg,
1.3 mmol) were added in portions under nitrogen at 0.degree. C. and
then stirred at 0.degree. C. for 1 hour. Acrylonitrile was
evaporated, the residue dissolved in DCM (25 ml) and extracted with
water (3.times.20 ml). Polymer precipitated between layers was
filtered off, the organic layer dried over K.sub.2CO.sub.3 and
evaporated in vacuum yielding 1.1 g of the title product.
[0394] MS (ES) m/z: [MH].sup.+=858.5
Example 75
2'-O-Acetyl-4''-O-(3-aminopropyl)-11-O-methyl-azithromycin
##STR00125##
[0396] To a solution of Example 74 (1.0 g, mmol) in glacial HOAc
(30 ml), PtO.sub.2 (0.2 g) was added and the reaction mixture was
hydrogenated in Parr apparatus at 5 bar for 18 hours. The catalyst
was filtered throught cellite and the solvent was evaporated in
vacuum, water (50 ml) was added, pH adjusted to 8.5 and extracted
with DCM (50 ml). The organic layer was evaporated in vacuum
yielding 0.9 g of the title product.
[0397] MS (ES) m/z: [MH].sup.+=862.4
[0398] .sup.13C-NMR(125 MHz, CDCl.sub.3) .delta.: 177.1, 169,0,
99.3, 94.3, 86.5, 85.0, 82.8, 77.2, 76.7, 74.0, 73.5, 72.5, 72.1,
71.4, 69.5, 66.6, 63.9, 62.6, 61.6, 60.8, 48.6, 44.5, 41.6, 41.5,
40.3, 39.2, 34.6, 34.0, 30.2, 27.2, 26.2, 22.0, 21.3, 21.1, 21.0,
18.1, 17.5, 14.4, 10.9, 8.6, 7.7.
Example 76
4''-O-(3-Aminopropyl)-11-O-methyl-azithromycin
##STR00126##
[0400] The compound of Example 75 (0.100 g, 0.11 mmol) was
dissolved in MeOH (40 ml) and the mixture was stirred at 50.degree.
C. for 18 hours. Methanol was evaporated under reduced pressure to
yield 95 mg of the title product.
[0401] MS (ES) m/z: [MH].sup.+820.3
[0402] .sup.13C-NMR(125 MHz, CDCl.sub.3) .delta.: 177.3, 101.8,
94.2, 86.7, 84.9, 82.5, 77.1, 76.6, 74.1, 73.4, 72.6, 71.9, 70.6,
69.7, 66.7, 64.6, 63.9, 61.7, 60.8, 48.7, 44.7, 42.1, 42.0, 40.3,
39.1, 34.7, 34.8, 30.1, 27.3, 26.2, 22.1, 21.6, 21.1, 21.0, 18.2,
17.6, 14.4, 10.9, 9.0, 7.7.
Examples 77 to 83
Intermediate:
2'-O-acetyl-4''-O-(imidazole-1-carbonyl)-6-O-methyl-9a-aza-9a-homoerythro-
mycin A
##STR00127##
[0404] A solution of
2'-O-acetyl-6-O-methyl-9a-aza-9a-homoerythromycin (4.00 g, 6.18
mmol) in dry toluene (50 ml) was stirred for 5 min. Triethylamine
(5.15 ml, 37.20 mmol) and CDI (2.43 ml, 13.67 mmol) were added and
the mixture stirred for 24 hours at room temperature. Second
portion of CDI (2.43 ml, 13.67 mmol) was added and stirring was
continued for further 48 hours. The reaction mixture was extracted
with saturated aqueous solution of NaHCO.sub.3 (1.times.50 ml,
2.times.30 ml). Combined aqueous layers were extracted with toluene
(2.times.20 ml). Combined toluene layers were dried over MgSO.sub.4
and evaporation under reduced pressure to gave 9.21 g of the title
product.
[0405] MS m/z: (ES): MH.sup.+=899.0
[0406] General Procedure for
4''-O-Carbamoyl-6-O-Methyl-9a-aza-9a-homoerythromycin A
Preparation
##STR00128##
TABLE-US-00014 Product R MS (ES. m/z) mass/mg purity % yield %
Example 77 ##STR00129## 906.2 [M + H].sup.+. calc. 905.2 22.2 99.5
43.8 Example 78 ##STR00130## 989.6 [M + H].sup.+. calc. 988.3 18.4
98.5 33.3 Example 79 ##STR00131## 975.2 [M + H].sup.+. calc. 974.3
21.8 99.0 40.0 Example 80 ##STR00132## 947.2 [M + H].sup.+. calc.
946.2 18.4 96.5 34.7 Example 81 ##STR00133## 920.4 [M + H].sup.+.
calc. 919.2 17.1 98.7 33.2 Example 82 ##STR00134## 892.2 [M +
H].sup.+. calc. 891.1 13.2 89.6 26.4 Example 83 ##STR00135## 878.2
[M + H].sup.+. calc. 877.1 23.9 100 50.7
[0407] Reaction mixture was stirred at 40.degree. C. for 48 hours
and then cooled to room temperature. Solvent was removed under
reduced pressure, oily product was dissolved in MeOH (3.0 ml) and
mixture was stirred at 40.degree. C. for 48 hours. After
evaporation of the solvent crude product was purified using
preparative HPLC (XTerra Prep RP18 column 5 .mu.m 19.times.100
mm).
Example 84
Diacetate Salt of Example 81
##STR00136##
[0409] To a solution of Example 81 (0.1 g, 0.11 mmol) in i-PrOH
(0.600 ml) acetic acid (0.0132 ml, 0.231 mmol) was added under
stirring in an ice bath. After addition of diisopropylether (3 ml)
and n-hexane (10 ml), solvent was evaporated under reduced pressure
to yield 0.100 g of the diacetate salt.
Example 85
Dihydrochloride Salt of Example 77
##STR00137##
[0411] Compound of Example 77 (0.100 g, 0.11 mmol) was dissolved in
i-PrOH (0.60 ml) and then HCl/i-PrOH (5 M, 0.0462 ml, 0.231 mmol)
was added. The hydrochloric salt was isolated by precipitation with
n-hexane (10 ml). After filtration 0.126 g of dihydrochlorie salt
was obtained.
Example 86
4''-O-[3-(2-Dimethylamino-ethylamino)-propionyl]-6-O-methyl-9a-aza-9a-homo-
erythromycin A
##STR00138##
[0413] A solution of
2'-O-acetyl-4''-O-propenoyl-6-O-methyl-9a-aza-9a-homoerythromycin A
(prepared according to WO 03/042228) (0.859 g, 1 mmol) and
N,N-dimethylethylamine (1.1 ml, 10 mmol) in MeOH (20 ml) was
stirred at 55.degree. C. for 18 hours. Methanol was evaporated and
residue purified by flash chromatography using
DCM/MeOH/NH.sub.4OH=90:9:1.5 as eluent to give 470 mg of the title
product.
[0414] MS (ES+) m/z: [MH].sup.+=905.5
Example 87
4''-O-(3-Diethylamino-propionyl)-11-O-(2-cyano-ethyl)-6-O-methyl-9a-aza-9a-
-homoerythromycin A
##STR00139##
[0416] Starting from 2'-O-acethyl protected compound of Example 8
and acrylonitrile, according to the procedure described for
Intermediate 2, Example 4, following deprotection of 2'-O-acetyl
group in methanol provides the title compound.
[0417] All compounds in this Example each have the substituent
bonded at the C-2 of the desosaminyl sugar in the S absolute
stereochemical configuration, and therefore, is in an anti
stereochemical configuration compared to the stereochemical
configurations of the substituents bonded at the C-1 and C-3 of the
desosaminyl sugar.
Example 88
4''-O-(3-Diethylamino-propionyl)-11-O-(3-aminopropyl)-6-O-methyl-9a-aza-9a-
-homoerythromycin A
[0418] Starting from the compound of Example 87 using the procedure
of Example 6 the title compound is obtained.
Example 89
3'-N-Demethyl-4''-O-propenoyl-azithromycin
##STR00140##
[0420] To a stirred solution of 4''-O-propenoyl-azithromycin (0.5
g, 0.62 mmol), obtained as described in international patent
application WO 03/042228 and NaOAc (0.23 g, 2.8 mmol) in MeOH (12.5
ml) solid iodine (0.16 g, 0.62 mmol) was added. The reaction
mixture was irradiated using 500 W halogen lamp for 2 hours, cooled
to room temperature, and the solvent evaporated. Solid residue was
dissolved in EtOAc (100 ml), filtrated, and the filtrate washed
with saturated NaHCO.sub.3 (25 ml) and saturated NaCl (25 ml).
Organic layer was dried over Na.sub.2SO.sub.4 and evaporated to
give the title product (0.4 g).
[0421] MS m/z: (ES):MH.sup.+=789.5.
Example 90
3'-N-Demethyl-4''-O-(3-diethylaminopropionyl)-azithromycin
##STR00141##
[0423] A solution of the compound of Example 89 (0.4 g, 0.51 mmol)
in diethyl amine (10 ml) was heated overnight at 40.degree. C.,
cooled to room temperature, and evaporated to dryness. Flash
cromatography of crude product on silica gel using
hexane/EtAc/diethyl amine =10:1:1 as eluent afforded the title
product (0.19 g).
[0424] MS m/z: (ES):MH.sup.+=862.8.
Example 91
3'-N-Demethyl-4''-O-propenoyl-6-O-methyl-erythromycin A
11,12-cyclic carbamate
##STR00142##
[0426] Starting from 4''-O-propenyl-6-O-methyl-erythromycin A
11,12-cyclic carbamate (0.25 g, 0.30 mmol), obtained as described
in international patent application WO 03/042228 and solid iodine
(0.08 g, 0.31 mmol) according to the procedure described for
Example 89 the title compound (0.21 g) was obtained.
[0427] MS m/z: (ES):MH.sup.+=813.6.
Example 92
3'-N-Demethyl-4''-O-(3-diethylaminopropionyl)-6-O-methyl-erythromycin
A 11,12-cyclic carbamate
##STR00143##
[0429] Starting from Example 91 (0.21 g, 0.26 mmol) and diethyl
amine (5 ml) according to the procedure described for the Example
90 the title compound (0.095 g) was obtained.
[0430] MS m/z: (ES):MH.sup.+=886.8.
Example 93
3'-N-Demethyl-4''-O-propenoyl-6-O-methyl-erythromycin A
##STR00144##
[0432] Starting from 4''-O-propenoyl-6-O-methyl-erythromycin A
(0.24 g, 0.30 mmol), obtained as described in international patent
application WO 03/042228 and solid iodine (0.08 g, 0.31 mmol)
according to the procedure described for Example 89 the title
compound (0.21 g) was obtained.
[0433] MS m/z: (ES): MH.sup.+=788.6.
Example 94
3'-N-Demethyl-4''-O-(3-diethylaminopropionyl)-6-O-methyl-erythromycin
A
##STR00145##
[0435] Starting from Example 93 (0.21 g, 0.27 mrnol) and diethyl
amine (5 ml) according to the procedure described for the Example
90 the title compound (0.15 g) was obtained.
[0436] MS m/z: (ES):MH.sup.+=861.7.
Example 95
4''-O-(3-diethylamino-propyl)-azithromycin 11,12-cyclic
carbonate
##STR00146## ##STR00147##
[0437] Intermediate 1 2'O-Acetyl-4''-O-(2-cyanoethyl)-azithromycin
11,12-cyclic carbonate
[0438] To the degassed solution of 20-acetyl-azithromycin
11,12-cyclic carbonate (10 g, 12.2 mmol) in acrylonitrile (250 ml)
were added t-BuOH (3.465 ml, 36 mmol) and NaH (528 mg, 13.2 mmol)
in portions at 0.degree. C. The reaction mixture was stirred for 12
hours allowing to reach room temperature. Acrylonitrile was
evaporated and the residue dissolved in DCM (50 ml), extracted with
water (3.times.50 ml). The organic layer was dried over
K.sub.2CO.sub.3 and evaporated in vacuum yielding the title product
(9.33 g).
[0439] MS m/z: (ES):MH.sup.+=870.6.
Intermediate 2 2'O-Acetyl-4''-O-(3-aminopropyl)-azithromycin
11,12-cyclic carbonate
[0440] Reduction of Intermediate 1 (3 g, 3.45 mmol) with PtO.sub.2
(1.0 g) in glacial HOAc (120 ml) at 5 bar for 18 hours gave the
title product (1.46 g).
[0441] MS m/z: (ES):MH.sup.+=875.0.
Intermediate 3 2'O-Acetyl-4''-O-(3-hydroxypropyl)-azithromycin
11,12-cyclic carbonate
[0442] To the solution of Intermediate 2 (5.0 g, 5.72 mmol) in 10%
water solution of HOAc (100 ml) was added NaNO.sub.2 (2.605 g,
37.75 mmol) in portions at 0.degree. C. After 3 hours, an
additional amount of NaNO.sub.2 (1.3 g, 18.84 mmol) was added. The
reaction mixture was left overnight at 4.degree. C. DCM was added
to the reaction mixture and the pH was adjusted to 10.7 by addition
of 20% NaOH. The layers were separated, organic layer dried over
K.sub.2CO.sub.3 and evaporated in vacuum yielding the title product
(4.70 g).
[0443] MS in/z: (ES):MH.sup.+=875.9
[0444] .sup.13C-NMR(75 MHz, CDCl.sub.3) .delta., ppm: 177.16,
170.36, 153.39, 100.11, 94.87, 87.24, 86.51, 84.83, 84.17, 77.54,
76.54, 74.69, 73.78, 73.44, 68.02, 67.41, 64.46, 63.42, 63.02,
61.62, 49.42, 45.07, 42.91, 42.45, 41.91, 34.99, 34.65, 32.44,
30.61, 27.06, 26.28, 22.26, 22.19, 22.09, 21.38, 18.38, 14.97,
13.86, 10.54, 10.01, 5.30.
Intermediate 4 4''-O-(3-hydroxypropyl)-azithromycin 11,12-cyclic
carbonate
[0445] Intermediate 3 (4.0 g, 4.6 mmol) was dissolved in MeOH (250
ml) and stirred overnight at 40.degree. C. Methanol was evaporated
under reduced pressure to give the title compound (2.3 g).
[0446] MS m/z: (ES):MH.sup.+=833.8.
4''-O-(3-diethylamino-propyl)-azithromycin 11,12-cyclic
carbonate
##STR00148##
[0448] To the solution of Intermediate 4, Example 95 (0.2 g, 0.24
mmol) in DCM (2 ml), Dess-Martin reagens (0.112 g, 0.264 mmol) was
added. The reaction mixture was stirred at room temperature
overnight; the aldehyde as white precipitate filtered off and
dissolved in DCM (15 ml). To the reaction mixture Et.sub.2NH (75
.mu.l, 0.72 mmol), NaBH(AOc).sub.3 (0.153 g, 0.72 mmol),
ZnCl.sub.2(0.033 g, 0.24 mmol) and sieves (4 .ANG.) were added,
stirred at room temperature for 2 hours and then filtered. The
filtrate was evaporated to afford yellow oily product (0.374 g),
which was purified by column chromatography
(DCM:MeOH:NH.sub.4OH=90:9:0.5) to afford the title product (0.117
g).
[0449] MS m/z: (ES):MH.sup.+=888.3
Example 96
4''-O-(3-diethylamino-propyl)-azithromycin
##STR00149##
[0451] To the solution of Example 95 (80 mg, 0.09 mmol) in MeOH (10
ml), K.sub.2CO.sub.3 (211 mg, 1.53 mmol) was added in water (3 ml).
The reaction mixture was stirred at room temperature overnight, the
MeOH was evaporated and the residue was extracted with DCM
(3.times.10 ml). Combined organic layers were dried over
K.sub.2CO.sub.3 and the solvent was evaporated. The product was
purified by column chromatography (DCM:MeOH:NH.sub.4OH=90:9:0.5) to
afford the title product (40 mg).
[0452] MS m/z: (ES):MH.sup.+=862.4
[0453] .sup.13C-NMR(75 MHz, CDCl.sub.3) .delta.: 178.94, 102.34,
94.85, 88.05, 83.23, 77.88, 77.46, 74.27, 73.81, 73.74, 73.68,
72.41, 71.02, 70.10, 67.88, 65.44, 64.70, 62.45, 49.56, 49.49,
46.70, 45.23, 42.32, 42.08, 40.37, 36.29, 35.44, 29.70, 29.09,
27.50, 26.80, 21.97, 21.79, 21.75, 21.30, 18.54, 16.22, 14.72,
11.27, 10.57, 9.16, 7.38.
Example 97
4''-O-{3-[4-(2-Diethylamino-ethyl)-piperazin-1-yl]-propyl}azithromycin
11,12-cyclic carbonate
##STR00150##
[0455] Starting from Intermediate 4 of Example 95 (0.2 g, 0.24
mmol), Dess-Martin reagent (0.112 g, 0.264 mmol) and
1-(2-diethylaminoethyl)piperazine (133 .mu.l, 0.72 mmol) were
combined and the title crude product (0.374 g) was obtained
according to the procedure for Example 95. The product was purified
by column chromatography (DCM:MeOH:NH.sub.4OH=90:9:1.5) to afford
the title product (37 mg).
[0456] MS m/z: (ES):MH.sup.+=1000.5
[0457] .sup.13C-NMR(75 MHz, CDCl.sub.3) .delta.:: 176.96, 153.41,
103.39, 95.86, 87.75, 85.50, 85.44, 78.37, 76.09, 73.87, 73.48,
72.77, 70.77, 68.48, 67.39, 64.96, 64.73, 60.93, 55.82, 55.14,
54.99, 53.61, 53.53, 53.04, 49.76, 49.69, 47.35, 45.74, 43.00,
41.83, 40.33, 35.82, 34.27, 28.89, 27.65, 26.78, 26.18, 22.06,
21.93, 21.59, 18.35, 15.15, 14.53, 14.11, 11.10, 10.80, 10.37,
5.90.
Example 98
4''-O-(3-diisopropylamino-propyl)-azithromycin 11,12-cyclic
carbonate
##STR00151##
[0459] Starting from Intermediate 4 of Example 95 (0.2 g, 0.24
mmol), Dess-Martin reagent (0.112 g, 0.264 mmol) and
diisopropylamine (102 .mu.l, 0.72 mmol) the title crude product
(0.374 g) was obtained according to the procedure for Example 95.
Product was purified by column chromatography
(DCM:MeOH:NH.sub.4OH=90:9:0.5) to afford the title product (28
mg).
[0460] MS m/z: (ES):MH.sup.+=916.7.
* * * * *